Methods for increasing thermogenic adipocytes

ABSTRACT

In certain aspects, the present invention provides compositions and methods for increasing thermogenic adipocytes (e.g., brown adipocytes or other UCP-1 expressing adipocytes) by administering an antagonist of an ActRIIB signaling pathway. Examples of such antagonists include ActRIIB polypeptides, anti-ActRIIB antibodies, anti-myostatin antibodies, anti-GDF3 antibodies, anti-Nodal, anti-activin, and anti-GDF11 antibodies. A variety of metabolic and other disorders may be treated by causing an increase in thermogenic adipocytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/438,810, filed Jun. 12, 2019 (now allowed), which is a continuationof U.S. application Ser. No. 15/703,007, filed Sep. 13, 2017 (nowabandoned), which is a continuation of U.S. application Ser. No.14/242,212, filed Apr. 1, 2014 (now U.S. Pat. No. 9,790,284), which is acontinuation of U.S. application Ser. No. 13/403,657, filed Feb. 23,2012 (now U.S. Pat. No. 8,703,694), which is a continuation of U.S.application Ser. No. 12/796,332, filed Jun. 8, 2010 (now U.S. Pat. No.8,178,488), which claims the benefit of U.S. Provisional ApplicationNos. 61/280,545, filed Nov. 3, 2009 (now expired), 61/276,422, filedSep. 10, 2009 (now expired), and 61/268,128, filed Jun. 8, 2009 (nowexpired). The specifications of each of the foregoing applications areincorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Feb. 23, 2021, is named1848179-050-106_Seq.txt, and is 35,349 bytes in size.

BACKGROUND OF THE INVENTION

Mammalian fat cells are traditionally classified as eitherenergy-storing white adipocytes or energy-expending brown adipocytes.Brown adipocytes express uncoupling protein-1 (UCP1), which convertsbiochemical energy to heat by uncoupling ATP production from themitochondrial proton gradient (Cannon et al., 2004, Physiol Rev84:277-359). Such thermogenesis serves to maintain body temperature incold environmental conditions or to promote energy balance in the faceof excess caloric intake. Underscoring the metabolic importance of brownfat, its genetic ablation in mice results in severe obesity accompaniedby insulin resistance, hyperglycemia, hyperlipidemia, andhypercholesterolemia (Lowell et al., 1993, Nature 366:740-742; Hamann etal., 1995, Diabetes 44:1266-1273; Hamann et al., 1996, Endocrinology137:21-29). Given the role of UCP-1 as an important uncoupling protein,adipocytes that express UCP-1 will have a thermogenic activity.

In humans, brown adipose tissue plays an important thermogenic role ininfants but shrinks during postnatal development and has historicallybeen dismissed as sparse and clinically unimportant in adults. However,recent findings have overturned this thinking and generated considerableinterest in the role(s) of brown adipose tissue during adulthood.Specifically, combined use of positron-emission tomography and computedtomography (PET-CT) to monitor tumor metastasis led to serendipidousdetection of highly active, putative brown fat depots in a substantialpercentage of adults (Nedergaard et al., 2007, Am J Physiol EndocrinolMetab 293:E444-E452). Subsequent studies have confirmed in healthyadults that these depots are indeed UCP1-expressing, functional brownfat (Virtanen et al., 2009, N Engl J Med 360:1518-1525), withbrown-adipose-tissue activity observed during cold exposure but notthermoneutral conditions in more than 90% of young men studied (vanMarken Lichtenbelt et al., 2009, N Engl J Med 360:1500-1508). Moreover,retrospective analysis of nearly two thousand PET-CT scans performed forvarious diagnostic reasons indicates that the amount of active brown fatis inversely correlated with body-mass index, a widely used measure ofoverall adiposity, raising the possibility of important beneficial rolesfor brown fat in adult human metabolism (Cypess et al., 2009, N Engl JMed 360:1509-1517). Less clear is the role of thermogenic adipocytes(e.g., brown adipocytes or other UCP-1 expressing adipocytes) that areinterspersed with white adipose tissue.

Given the important metabolic activities of thermogenic adipocytes,there is a need for agents that increase (e.g., by formation and/orincreased activity) thermogenic adipocytes in vivo.

SUMMARY OF THE INVENTION

In certain aspects, the present disclosure provides methods forincreasing thermogenic adipocytes in patients by using antagonists ofthe ActRIIB signaling pathway. Such antagonists may be, for example,soluble ActRIIB proteins (e.g., ActRIIB-Fc fusion proteins), antagoniststhat bind to ActRIIB or inhibit ActRIIB expression, and antagonists thatbind to or inhibit the expression of ligands that signal through ActRIIBand participate in the regulation of thermogenic adipocytes. Suchligands include myostatin (i.e., GDF8), GDF3, activins (e.g., activin A,B, C, or E), GDF11, and Nodal.

In certain aspects, the disclosure provides methods for increasingthermogenic adipocytes by administering to a patient in need thereof aneffective amount of an ActRIIB-related polypeptide. An ActRIIB-relatedpolypeptide may be an ActRIIB polypeptide (e.g., an ActRIIBextracellular domain or portion thereof) that binds to an ActRIIB ligandsuch as GDF3, GDF8, GDF11, activin or Nodal. Optionally, the ActRIIBpolypeptide binds to an ActRIIB ligand with a Kd less than 10 micromolaror less than 1 micromolar, 100, 10 or 1 nanomolar. A variety of suitableActRIIB polypeptides have been described in the following published PCTpatent applications, all of which are incorporated by reference herein:WO 00/43781, WO 04/039948, WO 06/012627, WO 07/053775, WO 08/097541, andWO 08/109167. Optionally, the ActRIIB polypeptide inhibits ActRIIBsignaling, such as intracellular signal transduction events triggered byan ActRIIB ligand. A soluble ActRIIB polypeptide for use in such apreparation may be any of those disclosed herein, such as a polypeptidehaving an amino acid sequence selected from SEQ ID NOs: 1, 2, 5, 6, 12,14 and 17, or having an amino acid sequence that is at least 80%, 85%,90%, 95%, 97% or 99% identical to an amino acid sequence selected fromSEQ ID NOs: 1, 2, 5, 6, 12, 14 and 17. A soluble ActRIIB polypeptide mayinclude a functional fragment of a natural ActRIIB polypeptide, such asone comprising at least 10, 20 or 30 amino acids of a sequence selectedfrom SEQ ID NOs: 1, 2, 5, 6, 12, 14 and 17 or a sequence of SEQ ID NO:1, lacking the C-terminal 1, 2, 3, 4, 5 or 10 to 15 amino acids andlacking 1, 2, 3, 4 or 5 amino acids at the N-terminus. Optionally,polypeptides will comprise a truncation relative to SEQ ID NO:1 ofbetween 2 and 5 amino acids at the N-terminus and no more than 3 aminoacids at the C-terminus. Another polypeptide is that presented as SEQ IDNO:12. A soluble ActRIIB polypeptide may include one, two, three, four,five or more alterations in the amino acid sequence (e.g., in theligand-binding domain) relative to a naturally occurring ActRIIBpolypeptide. The alteration in the amino acid sequence may, for example,alter glycosylation of the polypeptide when produced in a mammalian,insect or other eukaryotic cell or alter proteolytic cleavage of thepolypeptide relative to the naturally occurring ActRIIB polypeptide. Asoluble ActRIIB polypeptide may be a fusion protein that has, as onedomain, an ActRIIB polypeptide (e.g., a ligand-binding domain of anActRIIB or a variant thereof) and one or more additional domains thatprovide a desirable property, such as improved pharmacokinetics, easierpurification, targeting to particular tissues, etc. For example, adomain of a fusion protein may enhance one or more of in vivo stability,in vivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, multimerization of thefusion protein, and/or purification. A soluble ActRIIB fusion proteinmay include an immunoglobulin constant domain, such as an Fc domain(wild-type or mutant) or a serum albumin. In certain embodiments, anActRIIB-Fc fusion comprises a relatively unstructured linker positionedbetween the Fc domain and the extracellular ActRIIB domain. Thisunstructured linker may correspond to the roughly 15 amino acidunstructured region at the C-terminal end of the extracellular domain ofActRIIB (the “tail”), or it may be an artificial sequence of between 5and 15, 20, 30, 50 or more amino acids that are relatively free ofsecondary structure. A linker may be rich in glycine and prolineresidues and may, for example, contain repeating sequences ofthreonine/serine and glycines, e.g., TG₄ (SEQ ID NO: 18) or SG₄ repeats(SEQ ID NO: 19). A fusion protein may include a purificationsubsequence, such as an epitope tag, a FLAG tag, a polyhistidinesequence, and a GST fusion. Optionally, a soluble ActRIIB polypeptideincludes one or more modified amino acid residues selected from: aglycosylated amino acid, a PEGylated amino acid, a farnesylated aminoacid, an acetylated amino acid, a biotinylated amino acid, an amino acidconjugated to a lipid moiety, and an amino acid conjugated to an organicderivatizing agent. In general, it is preferable that an ActRIIB proteinbe expressed in a mammalian cell line that mediates suitably naturalglycosylation of the ActRIIB protein so as to diminish the likelihood ofan unfavorable immune response in a patient. Human and CHO cell lineshave been used successfully, and it is expected that other commonmammalian expression vectors will be useful.

In certain aspects, a compound disclosed herein may be formulated as apharmaceutical preparation. A pharmaceutical preparation may alsoinclude one or more additional compounds such as a compound that is usedto treat an ActRIIB-associated disorder. Preferably, a pharmaceuticalpreparation is substantially pyrogen free.

In certain aspects, the disclosure provides nucleic acids encoding asoluble ActRIIB polypeptide, which do not encode a complete ActRIIBpolypeptide. An isolated polynucleotide may comprise a coding sequencefor a soluble ActRIIB polypeptide, such as described above. For example,an isolated nucleic acid may include a sequence coding for anextracellular domain (e.g., ligand-binding domain) of an ActRIIB and asequence that would code for part or all of the transmembrane domainand/or the cytoplasmic domain of an ActRIIB, but for a stop codonpositioned within the transmembrane domain or the cytoplasmic domain, orpositioned between the extracellular domain and the transmembrane domainor cytoplasmic domain. For example, an isolated polynucleotide maycomprise a full-length ActRIIB polynucleotide sequence such as SEQ IDNO: 4, or a partially truncated version, said isolated polynucleotidefurther comprising a transcription termination codon at least sixhundred nucleotides before the 3′-terminus or otherwise positioned suchthat translation of the polynucleotide gives rise to an extracellulardomain optionally fused to a truncated portion of a full-length ActRIIB.Nucleic acids disclosed herein may be operably linked to a promoter forexpression, and the disclosure provides cells transformed with suchrecombinant polynucleotides. Preferably the cell is a mammalian cellsuch as a CHO cell.

In certain aspects, the disclosure provides methods for making a solubleActRIIB polypeptide. Such a method may include expressing any of thenucleic acids (e.g., SEQ ID NO: 3) disclosed herein in a suitable cell,such as a Chinese hamster ovary (CHO) cell. Such a method may comprise:a) culturing a cell under conditions suitable for expression of thesoluble ActRIIB polypeptide, wherein said cell is transformed with asoluble ActRIIB expression construct; and b) recovering the solubleActRIIB polypeptide so expressed. Soluble ActRIIB polypeptides may berecovered as crude, partially purified or highly purified fractionsusing any of the well known techniques for obtaining protein from cellcultures.

In certain aspects, increasing thermogenic adipocytes using a compounddescribed herein may be useful in the management of a variety ofdiseases in which management of metabolic activities is beneficial.Examples include management of obesity, decreasing the body fat contentor reducing the rate of increase in body fat content, and treating adisorder such as obesity, non-insulin dependent diabetes mellitus(NIDDM), type 2 diabetes, cardiovascular disease, cancer, hypertension,stroke, respiratory problems, dyslipidemia, lipodystrophy, consequencesof corticosteroid administration and gall bladder disease.

In certain aspects, a soluble ActRIIB polypeptide disclosed herein maybe used in a method for treating a subject having a disorder associatedwith muscle loss or insufficient muscle growth wherein such disorder isalso associated with a metabolic disorder, such as obesity,lipodystrophy, diabetes (e.g., type II diabetes), cachexia or otherdisorder described above. Such disorders include muscular dystrophy,sarcopenia and HIV (which may be associated with both a muscle wastingand a lipodystrophy).

In certain aspects, the disclosure provides methods for antagonizingactivity of an ActRIIB polypeptide or an ActRIIB ligand (e.g., GDF8,GDF11, activin, GDF3, and Nodal) in a cell. The methods comprisecontacting the cell with a soluble ActRIIB polypeptide. Optionally, theactivity of the ActRIIB polypeptide or the ActRIIB ligand is monitoredby a signaling transduction mediated by the ActRIIB/ActRIIB ligandcomplex, for example, by monitoring cell proliferation or the level ofUCP-1 expression. The cells of the methods include an osteoblast, achondrocyte, a myocyte, an adipocyte and a muscle cell.

In certain aspects, the disclosure provides uses of a soluble ActRIIBpolypeptide for making a medicament for the treatment of a disorder orcondition as described herein.

In certain aspects, the disclosure provides methods for increasingthermogenic adipocytes in a patient in need thereof, and such method maycomprise administering an effective amount of a compound selected fromthe group consisting of: a polypeptide comprising an amino acid sequencethat is at least 90% identical to the sequence of amino acids 29-109 ofSEQ ID NO:2 and a polypeptide encoded by a nucleic acid that hybridizesunder stringent hybridization conditions to a nucleic acid of SEQ IDNO:3. The polypeptide may be a fusion protein comprising a heterologousportion. The polypeptide may be a dimer. The polypeptide may be fused toa constant domain of an immunoglobulin. The polypeptide may be fused toan Fc portion of an immunoglobulin, such as an IgG1, IgG2, IgG3 or IgG4.The polypeptide may comprise an amino acid sequence that is at least80%, 90%, 93%, 95%, 97%, 98%, 99% or 100% identical to the sequence ofamino acids 29-109, 29-128, 29-131, 29-134, 25-109, 25-128, 25-131,25-134 or 20-134 of SEQ ID NO:2. The polypeptide may comprise an aminoacid sequence that is at least 80%, 90%, 93%, 95%, 97%, 98%, 99% or 100%identical to the sequence of amino acids of SEQ ID NO:5, 6, 12, 14 or17. A patient to be treated with such a compound may one having adisorder described herein, including, for example, a metabolic disorder(e.g., obesity, diabetes, metabolic syndrome, dyslipidemia orlipodystrophy) or a muscle disorder that is associated with a metabolicdisorder (e.g., some instances of sarcopenia). Administration of thecompound may promotes UCP-1 expression in adipocytes of the treatedpatient, optionally in the white adipose tissue.

In certain aspects, the disclosure provides methods for increasingthermogenic adipocytes in a patient in need thereof, the methodcomprising administering an effective amount of a compound that inhibitsthe ActRIIB signaling pathway, either by targeting ActRIIB or a ligandthat signals through ActRIIB. Examples of such compounds includeantagonists of ActRIIB; antagonists of myostatin (i.e., GDF-8);antagonists of activin (e.g., activin A, activin B, activin C, oractivin E); antagonists of GDF-11; antagonists of Nodal; and antagonistsof GDF3. Antagonists of each of the foregoing may be an antibody orother protein that specifically binds to and inhibits such target (e.g.,an antibody such as a monoclonal antibody, or a propeptide in the caseof myostatin and GDF3). Antagonists of the foregoing may also be acompound, such as a nucleic acid based compound (e.g., an antisense orRNAi nucleic acid) that inhibits the expression of ActRIIB or theligand. A patient to be treated with such a compound may one having adisorder described herein, including, for example, a metabolic disorder(e.g., obesity, diabetes, metabolic syndrome, dyslipidemia orlipodystrophy) or a muscle disorder that is associated with a metabolicdisorder (e.g., some instances of sarcopenia). Administration of thecompound may promotes UCP-1 expression in adipocytes of the treatedpatient, optionally in the white adipose tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Patent Office upon request andpayment of the necessary fee.

FIG. 1 shows the effect of ActRIIB (20-134)-hFc treatment for 60 days onuncoupling protein-1 (UCP1) mRNA levels in the epididymal fat pad ofmale mice fed a high-fat diet. RT-PCR data (in relative units, RU) aremeans±SEM; *, p<0.05 compared to vehicle. ActRIIB(20-134)-hFc caused anearly nine-fold increase in mRNA encoding this selective marker forbrown fat, thus indicating upregulation of thermogenic capability inbrown adipocytes distributed diffusely within this white fat depot.

FIG. 2 shows thermogenic histological changes induced within epididymalwhite adipose tissue by ActRIIB(25-131)-hFc treatment for 60 days inmice fed a high-fat diet. All microscopic images shown at the samemagnification. Hematoxylin and eosin (H&E) staining indicates theability of ActRIIB(25-131)-hFc to reduce lipid droplet size and induceclusters of multilocular adipocytes (arrows) characteristic of brownfat. Immunostaining of non-adjacent sections reveals widespreadcytoplasmic induction of UCP1 (green fluorescence) in both multilocularand unilocular adipocytes.

FIG. 3 shows the effect of ActRIIB(25-131)-hFc treatment for 60 days onUCP1 mRNA levels in epididymal white fat of mice fed a high-fat diet.RT-PCR data (in relative units, RU) are means±SEM; n=6-7 per group; *,p<0.05. ActRIIB(25-131)-hFc caused a 60-fold increase in mRNA encodingthis selective marker for brown fat, thus indicating upregulation ofthermogenic capability within this white fat depot.

FIG. 4 shows levels of mRNA encoding the sirtuin family member SIRT-1(silent information regulator two, homolog 1) in epididymal white fat ofmice as a function of diet and ActRIIB(25-131)-hFc treatment for 60days. RT-PCR data (in relative units, RU) are means±SEM; n=7 per group;*, p<0.05; NS=not significant. In mice fed a high-fat diet,ActRIIB(25-131)-hFc increased SIRT-1 mRNA levels by more than 70%,restoring them to levels not significantly different from those in micefed a standard diet.

FIG. 5 shows levels of mRNA encoding PGC-1α (peroxisomeproliferator-activated receptor gamma coactivator-1α) in epididymalwhite fat of mice as a function of diet and ActRIIB(25-131)-hFctreatment for 60 days. RT-PCR data (in relative units, RU) aremeans±SEM; n=6-7 per group; ***, p<0.001. In mice fed a high-fat diet,ActRIIB(25-131)-hFc increased PGC-1α mRNA levels by more than 250%,restoring them to levels not significantly different from those in micefed a standard diet.

FIG. 6 shows levels of mRNA encoding Foxo-1 (forkhead box-containing,protein O subfamily-1) in epididymal white fat of mice as a function ofdiet and ActRIIB(25-131)-hFc treatment for 60 days. RT-PCR data (inrelative units, RU) are means±SEM; n=7 per group; **, p<0.01. In micefed a high-fat diet, ActRIIB(25-131)-hFc increased Foxo-1 mRNA levels bymore than 90%, restoring them to levels not significantly different fromthose in mice fed a standard diet.

FIG. 7 shows levels of adiponectin mRNA in epididymal white fat of miceas a function of diet and ActRIIB(25-131)-hFc treatment for 60 days.RT-PCR data (in relative units, RU) are means±SEM; n=7 per group; *,p<0.05. In mice fed a high-fat diet, ActRIIB(25-131)-hFc increasedadiponectin mRNA levels by more than 60%, thus contributing to elevatedconcentrations of circulating adiponectin in these mice.

FIG. 8 shows serum levels of adiponectin in mice as a function of dietand ActRIIB(25-131)-hFc treatment for 60 days. ELISA measurements detectall main oligomeric isoforms (total adiponectin), and data aremeans±SEM; n=7-8 per group; **, p<0.01; ***, p<0.001. In mice fed ahigh-fat diet, ActRIIB(25-131)-hFc increased circulating adiponectinconcentrations by more than 75% to significantly exceed those instandard-diet controls.

FIG. 9 shows serum concentrations of insulin in mice as a function ofdiet and ActRIIB(25-131)-hFc treatment for 60 days. Data are means±SEM;n=7-8 per group; **, p<0.01. In mice fed a high-fat diet,ActRIIB(25-131)-hFc normalized insulin concentrations to levels observedin standard-diet controls.

FIG. 10 shows photographs of bilateral pairs of interscapular brown fatdepots as a function of diet and ActRIIB(25-131)-mFc treatment for 60days. High-fat diet increased the size and lightened the color of thedepots, whereas ActRIIB(25-131)-mFc largely reversed these changes.

FIG. 11 depicts the effect of ActRIIB(25-131)-mFc treatment for 60 dayson the mass of interscapular brown fat in mice fed a high-fat diet. Dataare means±SEM for combined left and right depots; ***, p<0.001.ActRIIB(25-131)-mFc reversed the effect of high-fat diet on the mass ofthis brown fat depot.

FIG. 12 depicts the effect of ActRIIB(25-131)-mFc treatment for 60 dayson the density of interscapular brown fat in mice fed a high-fat diet,as determined by micro-computed tomography (microCT). Data (means±SEM)are expressed in standardized units based on a positive value for thebone mineral hydroxyapatite (HA) and a value of zero for water;therefore, fat values are negative, with values for white fat typicallyclose to −120. **, p<0.01. ActRIIB(25-131)-mFc completely reversed theeffect of high-fat diet on the density of this brown fat depot.

FIG. 13 shows the full amino acid sequence of ActRIIB(25-131)-hFc (SEQID NO:14). The TPA leader (residues 1-22) and truncated ActRIIBextracellular domain (native residues 25-131) are each underlined.Highlighted is the glutamate revealed by sequencing to be the N-terminalamino acid of the mature fusion protein.

FIGS. 14A and 14B show a nucleotide sequence encodingActRIIB(25-131)-hFc (the coding strand, SEQ ID NO: 15, is shown at topand the complement, SEQ ID NO: 16, is shown at bottom 3′-5′). Sequencesencoding the TPA leader (nucleotides 1-66) and ActRIIB extracellulardomain (nucleotides 73-396) are underlined. The corresponding amino acidsequence for ActRIIB(25-131) is also shown.

DETAILED DESCRIPTION 1. Overview

Mammalian fat cells can be classified as either energy-storing whiteadipocytes or energy-expending brown adipocytes. Uncoupling protein-1(UCP1), which converts biochemical energy to heat by uncoupling ATPproduction from the mitochondrial proton gradient, is widely consideredto be the definitive functional marker for brown adipocytes. Adipocytesexpressing UCP-1 are referred to herein as “thermogenic adipocytes”.Genetic ablation of brown adipose tissue in mice leads to extremeobesity (Lowell et al., 1993, Nature 366:740-742), and selectiveablation of UCP1 prevents the thermogenic and anti-obesity responses toβ₃-adrenergic stimulation in mice (Inokuma et al., 2006, Am J PhysiolEndocrinol Metab 290:E1014-E1021), confirming that UCP1 is a criticalmolecule in the regulation of energy expenditure and adiposity (Kozak etal., 2008, Int J Obes 32:S32-S38).

In mammals ranging from rodents to humans, brown adipocytes occur indiscrete depots of brown adipose tissue that are most prominentneonatally, consistent with the thermal challenges to survival at thisage. Recent findings indicate that these brown fat depots persist withthermogenic capability during adulthood in humans (Nedergaard et al.,2007, Am J Physiol Endocrinol Metab 293:E444-E452; van MarkenLichtenbelt et al., 2009, N Engl J Med 360:1500-1508; Cypess et al.,2009, N Engl J Med 360:1509-1517), raising the possibility that suchtissue might be activated exogenously for therapeutic benefit.Intriguingly, considerable numbers of brown adipocytes also occurtransiently within some ‘white’ fat depots during early postnataldevelopment (Xue et al., 2007, J Lipid Res 48:41-51) and can reappear inwhite fat depots under certain conditions in adulthood (Cousin et al.,1992, J Cell Sci 103:931-942). Even in humans, limited evidence suggeststhat brown adipocytes are inducible in white fat depots during adulthood(Lean et al., 1986, Int J Obes 10:219-227). Thus, there is also thepossibility that ‘diffuse’ thermogenic adipocytes could be induced intraditional white fat depots for therapeutic benefit. Traditional depotsof white adipose tissue, in fact, display a degree of cellularremodeling, or phenotypic plasticity, not observed in discrete brown fatdepots (Prunet-Marcassus et al., 2006, Exp Cell Res 312:727-736).

As described in the Examples, an ActRIIB-Fc fusion protein can be usedto increase UCP-1 signaling in fat depots of mice fed a high fat diet.Therefore, ActRIIB-derived agents and other compounds that inhibitActRIIB signaling can be used to increase the number and/or activity ofthermogenic adipocytes. Ligands that bind to ActRIIB which areimplicated in the regulation of thermogenic adipocytes include theactivins (e.g., activin A, activin B, activin C, and activin E),myostatin (i.e., GDF-8), GDF-3, GDF-11, and Nodal. In certain aspects,the present invention relates to ActRIIB polypeptides. As used herein,the term “ActRIIB” refers to a family of activin receptor type IIB(ActRIIB) proteins and ActRIIB-related proteins, derived from anyspecies. Members of the ActRIIB family are generally all transmembraneproteins, composed of a ligand-binding extracellular domain withcysteine-rich region, a transmembrane domain, and a cytoplasmic domainwith predicted serine/threonine kinase specificity. The human ActRIIBprecursor has the following amino acid sequence, with the signal peptideunderlined, the extracellular domain indicated in bold, and thepotential N-linked glycosylation sites boxed (SEQ ID NO: 2) (NM_001106,512 aa).

AGGPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQHPSLEELQEVVVHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSLIRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI

The above wild type sequence, including the native leader, is usedthroughout this disclosure as the base sequence for numbering the aminoacids of any of the various truncations, mature forms and variants ofActRIIB. The term “ActRIIB polypeptide” is used to refer to polypeptidescomprising any naturally occurring polypeptide of an ActRIIB familymember as well as any variants thereof (including mutants, fragments,fusions, and peptidomimetic forms) that retain a useful activity. Forexample, ActRIIB polypeptides include polypeptides derived from thesequence of any known ActRIIB having a sequence at least about 80%identical to the sequence of an ActRIIB polypeptide, and preferably atleast 85%, 90%, 95%, 97%, 99% or greater identity.

In a specific embodiment, the invention relates to soluble ActRIIBpolypeptides. As described herein, the term “soluble ActRIIBpolypeptide” generally refers to polypeptides comprising anextracellular domain of an ActRIIB protein. The term “soluble ActRIIBpolypeptide,” as used herein, includes any naturally occurringextracellular domain of an ActRIIB protein as well as any variantsthereof (including mutants, fragments and peptidomimetic forms) thatretain a useful activity. For example, the extracellular domain of anActRIIB protein binds to a ligand and is generally soluble. Thefollowing is an example of a soluble ActRIIB polypeptide (SEQ ID NO: 1)(116 aa).

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHL PEAGGPEVTYEPPPTAPT

Other examples of soluble ActRIIB polypeptides comprise a signalsequence in addition to the extracellular domain of an ActRIIB protein(see Example 1). The signal sequence can be a native signal sequence ofan ActRIIB, or a signal sequence from another protein, such as a tissueplasminogen activator (TPA) signal sequence or a honey bee mellitin(HBM) signal sequence.

Two related type II receptors, ActRIIA and ActRIIB, have been identifiedas the type II receptors for activins (Mathews and Vale, 1991, Cell65:973-982; Attisano et al., 1992, Cell 68: 97-108) as well as a varietyof other BMPs and GDFs. Besides activins, ActRIIA and ActRIIB canbiochemically interact with several other TGF-β family proteins,including BMP7, Nodal, GDF8, and GDF11 (Yamashita et al., 1995, J. CellBiol. 130:217-226; Lee and McPherron, 2001, Proc. Natl. Acad. Sci.98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7: 949-957; Oh et al.,2002, Genes Dev. 16:2749-54). In certain embodiments, the presentinvention relates to antagonizing a ligand of ActRIIB receptors (alsoreferred to as an ActRIIB ligand) with a subject ActRIIB polypeptide(e.g., a soluble ActRIIB polypeptide). Thus, compositions and methods ofthe present invention are useful for treating disorders associated withabnormal activity of one or more ligands of ActRIIB receptors. Exemplaryligands of ActRIIB receptors include some TGF-β family members, such asactivin (e.g., activin A, activin B, activin C, and activin E), GDF3,Nodal, GDF8, and GDF11.

Activins are dimeric polypeptide growth factors and belong to theTGF-beta superfamily. There are three activins (A, B, and AB) that arehomo/heterodimers of two closely related β subunits (β_(A)β_(A),β_(B)β_(B), and β_(A)β_(B)). In the TGF-beta superfamily, activins areunique and multifunctional factors that can stimulate hormone productionin ovarian and placental cells, support neuronal cell survival,influence cell-cycle progress positively or negatively depending on celltype, and induce mesodermal differentiation at least in amphibianembryos (DePaolo et al., 1991, Proc Soc Ep Biol Med. 198:500-512; Dysonet al., 1997, Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol.55:953-963). Moreover, erythroid differentiation factor (EDF) isolatedfrom the stimulated human monocytic leukemic cells was found to beidentical to activin A (Murata et al., 1988, PNAS, 85:2434). It wassuggested that activin A acts as a natural regulator of erythropoiesisin the bone marrow. In several tissues, activin signaling is antagonizedby its related heterodimer, inhibin. For example, during the release offollicle-stimulating hormone (FSH) from the pituitary, activin promotesFSH secretion and synthesis, while inhibin prevents FSH secretion andsynthesis. Other proteins that may regulate activin bioactivity and/orbind to activin include follistatin (FS), follistatin-related protein(FSRP), α₂-macroglobulin, Cerberus, and endoglin, which are describedbelow.

Bone morphogenetic protein 7 (BMP7), also called osteogenic protein-1(OP-1), is well known to induce cartilage and bone formation. Inaddition, BMP7 regulates a wide array of physiological processes.Notably, BMP7 has recently been identified as a key promoter of brownadipocyte differentiation (Tseng et al., 2008, Nature 454:1000-1004). Inthis study, genetic ablation of BMP7 led to scarcity of brown fat andnearly complete absence of UCP1 in murine embryos. Moreover,upregulation of BMP7 expression in mice by adenovirus administrationincreased brown fat mass and energy expenditure. Therefore, theliterature would suggest that an antagonist of BMP7 such as an ActRIIBpolypeptide or anti-ActRIIB antibody would not be expected to promoteUCP1 expression, brown adipocyte formation, and/or brown adipocyteactivity. Like activin, BMP7 binds to type II receptors, ActRIIA andActRIIB. However, BMP7 and activin recruit distinct type I receptorsinto heteromeric receptor complexes. The major BMP7 type I receptorobserved was ALK2, while activin bound exclusively to ALK4 (ActRIIB).BMP7 and activin elicited distinct biological responses and activateddifferent Smad pathways (Macias-Silva et al., 1998, J Biol Chem.273:25628-36).

Growth-and-Differentiation Factor-3 (GDF3), also known as Vg1-related 2,plays an important role in embryonic development and has also beenimplicated in adipogenesis during adulthood. In brief, expression ofGDF3 in white adipose tissue is correlated with body mass or obesity(Weisberg et al., 2003, J Clin Invest 112:1796-1808), andadenovirus-mediated overexpression of GDF3 exaggerates the increase inadiposity observed under high-fat dietary conditions in wildtype mice(Wang et al., 2004, Biochem Biophys Res Commun 321:1024-1031).Importantly, mice with genetic ablation of GDF3 are healthy andessentially normal when maintained on a standard diet but are protectedfrom obesity, and display an increased basal metabolic rate, whenmaintained on a high-fat diet (Shen et al., 2009, Mol Endocrinol23:113-123). Taken together, these findings implicate GDF3 specificallyin diet-induced obesity and more generally in the regulation ofadiposity.

Nodal proteins have functions in mesoderm and endoderm induction andformation, as well as subsequent organization of axial structures suchas heart and stomach in early embryogenesis. It has been demonstratedthat dorsal tissue in a developing vertebrate embryo contributespredominantly to the axial structures of the notochord and pre-chordalplate while it recruits surrounding cells to form non-axial embryonicstructures. Nodal appears to signal through both type I and type IIreceptors and intracellular effectors known as Smad proteins. Recentstudies support the idea that ActRIIA and ActRIIB serve as type IIreceptors for Nodal (Sakuma et al., Genes Cells. 2002, 7:401-12). It issuggested that Nodal ligands interact with their co-factors (e.g.,cripto) to activate activin type I and type II receptors, whichphosphorylate Smad2. Nodal proteins are implicated in many eventscritical to the early vertebrate embryo, including mesoderm formation,anterior patterning, and left-right axis specification. Experimentalevidence has demonstrated that Nodal signaling activates pAR3-Lux, aluciferase reporter previously shown to respond specifically to activinand TGF-beta. However, Nodal is unable to induce pTlx2-Lux, a reporterspecifically responsive to bone morphogenetic proteins. Recent resultsprovide direct biochemical evidence that Nodal signaling is mediated byboth activin-TGF-beta pathway Smads, Smad2 and Smad3. Further evidencehas shown that the extracellular cripto protein is required for Nodalsignaling, making it distinct from activin or TGF-beta signaling.

Growth and Differentiation Factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highlyexpressed in the developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of the skeletal muscle (McPherron et al., Nature, 1997,387:83-90). Similar increases in skeletal muscle mass are evident innaturally occurring mutations of GDF8 in cattle (Ashmore et al., 1974,Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci., 1994,38:752-757; McPherron and Lee, Proc. Natl. Acad. Sci. USA, 1997,94:12457-12461; and Kambadur et al., Genome Res., 1997, 7:910-915) and,strikingly, in humans (Schuelke et al., N Engl J Med 2004; 350:2682-8).Studies have also shown that muscle wasting associated withHIV-infection in humans is accompanied by increases in GDF8 proteinexpression (Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). Inaddition, GDF8 can modulate the production of muscle-specific enzymes(e.g., creatine kinase) and modulate myoblast cell proliferation (WO00/43781). The GDF8 propeptide can noncovalently bind to the mature GDF8domain dimer, inactivating its biological activity (Miyazono et al.(1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J. Biol.Chem., 263; 7646-7654; and Brown et al. (1990) Growth Factors, 3:35-43). Other proteins which bind to GDF8 or structurally relatedproteins and inhibit their biological activity include follistatin, andpotentially, follistatin-related proteins (Gamer et al. (1999) Dev.Biol., 208: 222-232).

Growth and Differentiation Factor-11 (GDF11), also known as BMP11, is asecreted protein (McPherron et al., 1999, Nat. Genet. 22: 260-264).GDF11 is expressed in the tail bud, limb bud, maxillary and mandibulararches, and dorsal root ganglia during mouse development (Nakashima etal., 1999, Mech. Dev. 80: 185-189). GDF11 plays a unique role inpatterning both mesodermal and neural tissues (Gamer et al., 1999, DevBiol., 208:222-32). GDF11 was shown to be a negative regulator ofchondrogenesis and myogenesis in developing chick limb (Gamer et al.,2001, Dev Biol. 229:407-20). The expression of GDF11 in muscle alsosuggests its role in regulating muscle growth in a similar way to GDF8.In addition, the expression of GDF11 in brain suggests that GDF11 mayalso possess activities that relate to the function of the nervoussystem. Interestingly, GDF11 was found to inhibit neurogenesis in theolfactory epithelium (Wu et al., 2003, Neuron. 37:197-207). Hence, GDF11may have in vitro and in vivo applications in the treatment of diseasessuch as muscle diseases and neurodegenerative diseases (e.g.,amyotrophic lateral sclerosis).

In certain aspects, the present invention relates to the use of certainActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) to antagonizethe signaling of ActRIIB ligands generally, in any process associatedwith ActRIIB activity. Optionally, ActRIIB polypeptides of the inventionmay antagonize one or more ligands of ActRIIB receptors, such as activin(e.g., activin A, activin B, activin C, and activin E), GDF3, Nodal,GDF8, and GDF11, and may therefore be useful in the treatment ofadditional disorders.

Therefore, the present disclosure contemplates using ActRIIBpolypeptides and antagonists of ActRIIB or ActRIIB ligands in treatingor preventing diseases or conditions that are related to the activitiesof thermogenic adipocytes. ActRIIB or ActRIIB ligands are involved inthe regulation of many critical biological processes. Examples of suchmetabolic disorders or conditions include, but are not limited to,metabolic syndrome (also known as syndrome X), diabetes, impairedglucose tolerance, impaired fasting glucose, elevated plasma insulinconcentrations and insulin resistance, dyslipidemias, hyperlipidemia,overeating and bulimia, cancers of the colon, prostate, breast,endometrium, and kidney, osteoarthritis, obstructive sleep apnea,cholelithiasis, gallstones, hypertension, heart disease, abnormal heartrhythms and arrythmias, myocardial infarction, congestive heart failure,coronary heart disease, coronary artery disease, angina pectoris, suddendeath, polycystic ovarian disease, craniopharyngioma, the Prader-Willisyndrome, Frohlich's syndrome, GH-deficient subjects, normal variantshort stature, Turner's syndrome, and other pathological conditionsshowing reduced metabolic activity or a decrease in resting energyexpenditure as a percentage of total fat-free mass, e.g., children withacute lymphoblastic leukemia. Further examples are sexual andreproductive dysfunction (such as infertility), hypogonadism in malesand hirsutism in females, gastrointestinal motility disorders (such asobesity-related gastro-esophageal reflux, respiratory disorders (such asobesity-hypoventilation syndrome or Pickwickian syndrome),cardiovascular disorders, cerebral infarction, cerebral thrombosis,transient ischemic attack, inflammation (such as systemic inflammationof the vasculature), arteriosclerosis, hypercholesterolemia,hyperuricacidemia, fatty liver, gout, gallbladder disease, orthopedicdisorders, and lower back pain. These disorders and conditions arediscussed below under “Exemplary Therapeutic Uses.”

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

The term “diabetes”, as used herein, refers to non-insulin-dependentdiabetes mellitus (NIDDM, also known as type II diabetes). Type Idiabetes, or insulin-dependent diabetes mellitus (IDDM), is the resultof an absolute deficiency of insulin, the hormone which regulatesglucose utilization. Type II diabetes, or insulin-dependent diabetes(i.e., non-insulin-dependent diabetes mellitus), often occurs in theface of normal, or even elevated, levels of insulin and appears to bethe result of the inability of tissues to respond appropriately toinsulin. Most type II diabetics are also obese.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

“Obesity” is a condition in which there is an excess of body fat. Theoperational definition of obesity is based on the body mass index (BMI),calculated as body weight per height in meters squared (kg/m²).“Obesity” refers to a condition that is diagnosed as such by aphysician. One standard grading system is described as follows forpatients of generally European, African, Native American or Indiandescent, and an alternative system is often used for Asian patients.According to this system, obesity is defined as an otherwise healthysubject that has a BMI greater than or equal to 30 kg/m², or a conditionwhereby a subject with at least one co-morbidity has a BMI greater thanor equal to 27 kg/m².

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

2. ActRIIB Polypeptides

In certain aspects, the invention relates to ActRIIB variantpolypeptides (e.g., soluble ActRIIB polypeptides). Optionally, thefragments, functional variants, and modified forms have similar or thesame biological activities of their corresponding wild-type ActRIIBpolypeptides. For example, an ActRIIB variant of the invention may bindto and inhibit function of an ActRIIB ligand (e.g., activin A, activinAB, activin B, activin C, activin E GDF3, Nodal, GDF8, or GDF11).Optionally, an ActRIIB polypeptide modulates growth of tissues such asfat, muscle, bone, or cartilage. Examples of ActRIIB polypeptidesinclude human ActRIIB precursor polypeptide (SEQ ID NO: 2), and solublehuman ActRIIB polypeptides (e.g., SEQ ID NOs: 1, 5, 6, 12, 14, and 17).

The disclosure identifies functionally active portions and variants ofActRIIB. Applicants have ascertained that an Fc fusion protein havingthe sequence disclosed by Hilden et al. (Blood. 1994 Apr. 15;83(8):2163-70), which has an Alanine at the position corresponding toamino acid 64 of SEQ ID NO: 2 (A64), has a relatively low affinity foractivin and GDF-11. By contrast, the same Fc fusion protein with anArginine at position 64 (R64) has an affinity for activin and GDF-11 inthe low nanomolar to high picomolar range. Therefore, a sequence with anR64 is used as the wild-type reference sequence for human ActRIIB inthis disclosure.

Attisano et al. (Cell. 1992 Jan. 10; 68(1):97-108) showed that adeletion of the proline knot at the C-terminus of the extracellulardomain of ActRIIB reduced the affinity of the receptor for activin. AnActRIIB-Fc fusion protein containing amino acids 20-119 of SEQ ID NO:2,“ActRIIB(20-119)-Fc” has reduced binding to GDF-11 and activin relativeto an ActRIIB(20-134)-Fc, which includes the proline knot region and thecomplete juxtamembrane domain. However, an ActRIIB(20-129)-Fc proteinretains similar but somewhat reduced activity relative to the wild type,even though the proline knot region is disrupted. Thus, ActRIIBextracellular domains that stop at amino acid 134, 133, 132, 131, 130and 129 are all expected to be active, but constructs stopping at 134 or133 may be most active. Similarly, mutations at any of residues 129-134are not expected to alter ligand binding affinity by large margins. Insupport of this, mutations of P129 and P130 do not substantiallydecrease ligand binding. Therefore, an ActRIIB-Fc fusion protein may endas early as amino acid 109 (the final cysteine), however, forms endingat or between 109 and 119 are expected to have reduced ligand binding.Amino acid 119 is poorly conserved and so is readily altered ortruncated. Forms ending at 128 or later retain ligand binding activity.Forms ending at or between 119 and 127 will have an intermediate bindingability. Any of these forms may be desirable to use, depending on theclinical or experimental setting.

At the N-terminus of ActRIIB, it is expected that a protein beginning atamino acid 29 or before will retain ligand binding activity. Amino acid29 represents the initial cysteine. An alanine to asparagine mutation atposition 24 introduces an N-linked glycosylation sequence withoutsubstantially affecting ligand binding. This confirms that mutations inthe region between the signal cleavage peptide and the cysteinecross-linked region, corresponding to amino acids 20-29 are welltolerated. In particular, constructs beginning at position 20, 21, 22,23 and 24 will retain activity, and constructs beginning at positions25, 26, 27, 28 and 29 are also expected to retain activity.

Taken together, an active portion of ActRIIB comprises amino acids29-109 of SEQ ID NO:2, and constructs may, for example, begin at aresidue corresponding to amino acids 20-29 and end at a positioncorresponding to amino acids 109-134. Other examples include constructsthat begin at a position from 20-29 or 21-29 and end at a position from119-134, 119-133 or 129-134, 129-133. Other examples include constructsthat begin at a position from 20-24 (or 21-24, or 22-25) and end at aposition from 109-134 (or 109-133), 119-134 (or 119-133) or 129-134 (or129-133). Variants within these ranges are also contemplated,particularly those having at least 80%, 85%, 90%, 95% or 99% identity tothe corresponding portion of SEQ ID NO:2.

The disclosure includes the results of an analysis of composite ActRIIBstructures demonstrating that the ligand binding pocket is defined byresidues Y31, N33, N35, L38 through T41, E47, E50, Q53 through K55, L57,H58, Y60, S62, K74, W78 through N83, Y85, R87, A92, and E94 throughF101. At these positions, it is expected that conservative mutationswill be tolerated, although a K74A mutation is well-tolerated, as areR40A, K55A, F82A and mutations at position L79. R40 is a K in Xenopus,indicating that basic amino acids at this position will be tolerated.Q53 is R in bovine ActRIIB and K in Xenopus ActRIIB, and therefore aminoacids including R, K, Q, N and H will be tolerated at this position.Thus, a general formula for an active ActRIIB variant protein is onethat comprises amino acids 29-109, but optionally beginning at aposition ranging from 20-24 or 22-25 and ending at a position rangingfrom 129-134, and comprising no more than 1, 2, 5, 10 or 15 conservativeamino acid changes in the ligand binding pocket, and zero, one or morenon-conservative alterations at positions 40, 53, 55, 74, 79 and/or 82in the ligand binding pocket. Such a protein may retain greater than80%, 90%, 95% or 99% sequence identity to the sequence of amino acids29-109 of SEQ ID NO:2. Sites outside the binding pocket, at whichvariability may be particularly well tolerated, include the amino andcarboxy termini of the extracellular domain (as noted above), andpositions 42-46 and 65-73. An asparagine to alanine alteration atposition 65 (N65A) actually improves ligand binding in the A64background, and is thus expected to have no detrimental effect on ligandbinding in the R64 background. This change probably eliminatesglycosylation at N65 in the A64 background, thus demonstrating that asignificant change in this region is likely to be tolerated. While anR64A change is poorly tolerated, R64K is well-tolerated, and thusanother basic residue, such as H may be tolerated at position 64.

ActRIIB is well-conserved across nearly all vertebrates, with largestretches of the extracellular domain conserved completely. Many of theligands that bind to ActRIIB are also highly conserved. Accordingly,comparisons of ActRIIB sequences from various vertebrate organismsprovide insights into residues that may be altered. Therefore, anactive, human ActRIIB variant may include one or more amino acids atcorresponding positions from the sequence of another vertebrate ActRIIB,or may include a residue that is similar to that in the human or othervertebrate sequence. The following examples illustrate this approach todefining an active ActRIIB variant. L46 is a valine in Xenopus ActRIIB,and so this position may be altered, and optionally may be altered toanother hydrophobic residue, such as V, I or F, or a non-polar residuesuch as A. E52 is a K in Xenopus, indicating that this site may betolerant of a wide variety of changes, including polar residues, such asE, D, K, R, H, S, T, P, G, Y and probably A. T93 is a K in Xenopus,indicating that a wide structural variation is tolerated at thisposition, with polar residues favored, such as S, K, R, E, D, H, G, P, Gand Y. F108 is a Y in Xenopus, and therefore Y or other hydrophobicgroup, such as I, V or L should be tolerated. E111 is K in Xenopus,indicating that charged residues will be tolerated at this position,including D, R, K and H, as well as Q and N. R112 is K in Xenopus,indicating that basic residues are tolerated at this position, includingR and H. A at position 119 is relatively poorly conserved, and appearsas P in rodents and V in Xenopus, thus essentially any amino acid shouldbe tolerated at this position.

The disclosure demonstrates that the addition of a further N-linkedglycosylation site (N-X-S/T) increases the serum half-life of anActRIIB-Fc fusion protein, relative to the ActRIIB(R64)-Fc form. Byintroducing an asparagine at position 24 (A24N construct), an NXTsequence is created that confers a longer half-life. Other NX(T/S)sequences are found at 42-44 (NQS) and 65-67 (NSS), although the lattermay not be efficiently glycosylated with the R at position 64. N-X-S/Tsequences may be generally introduced at positions outside the ligandbinding pocket. Particularly suitable sites for the introduction ofnon-endogenous N-X-S/T sequences include amino acids 20-29, 20-24,22-25, 109-134, 120-134 or 129-134. N-X-S/T sequences may also beintroduced into the linker between the ActRIIB sequence and the Fc orother fusion component. Such a site may be introduced with minimaleffort by introducing an N in the correct position with respect to apre-existing S or T, or by introducing an S or T at a positioncorresponding to a pre-existing N. Thus, desirable alterations thatwould create an N-linked glycosylation site are: A24N, R64N, S67N(possibly combined with an N65A alteration), E106N, R112N, G120N, E123N,P129N, A132N, R112S and R112T. Any S that is predicted to beglycosylated may be altered to a T without creating an immunogenic site,because of the protection afforded by the glycosylation. Likewise, any Tthat is predicted to be glycosylated may be altered to an S. Thus thealterations S67T and S44T are contemplated. Likewise, in an A24Nvariant, an S26T alteration may be used. Accordingly, an ActRIIB variantmay include one or more additional, non-endogenous N-linkedglycosylation consensus sequences.

Position L79 may be altered to confer altered activin-myostatin (GDF-11)binding properties. L79A or L79P reduces GDF-11 binding to a greaterextent than activin binding. L79E or L79D retains GDF-11 binding.Remarkably, the L79E and L79D variants have greatly reduced activinbinding. In vivo experiments indicate that these non-activin receptorsretain significant ability to increase muscle mass but show decreasedeffects on other tissues. These data demonstrate the desirability andfeasibility for obtaining polypeptides with reduced effects on activin.

The variations described may be combined in various ways. Additionally,the results of mutagenesis program described herein indicate that thereare amino acid positions in ActRIIb that are often beneficial toconserve. These include position 64 (basic amino acid), position 80(acidic or hydrophobic amino acid), position 78 (hydrophobic, andparticularly tryptophan), position 37 (acidic, and particularly asparticor glutamic acid), position 56 (basic amino acid), position 60(hydrophobic amino acid, particularly phenylalanine or tyrosine). Thus,in each of the variants disclosed herein, the disclosure provides aframework of amino acids that may be conserved. Other positions that maybe desirable to conserve are as follows: position 52 (acidic aminoacid), position 55 (basic amino acid), position 81 (acidic), 98 (polaror charged, particularly E, D, R or K).

In certain embodiments, isolated fragments of the ActRIIB polypeptidescan be obtained by screening polypeptides recombinantly produced fromthe corresponding fragment of the nucleic acid encoding an ActRIIBpolypeptide (e.g., SEQ ID NOs: 3 and 4). In addition, fragments can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Thefragments can be produced (recombinantly or by chemical synthesis) andtested to identify those peptidyl fragments that can function, forexample, as antagonists (inhibitors) or agonists (activators) of anActRIIB protein or an ActRIIB ligand.

In certain embodiments, a functional variant of the ActRIIB polypeptideshas an amino acid sequence that is at least 75% identical to an aminoacid sequence selected from SEQ ID NOs: 1, 2, 5, 6, 12, 14, and 17. Incertain cases, the functional variant has an amino acid sequence atleast 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an aminoacid sequence selected from SEQ ID NOs: 1, 2, 5, 6, 12, 14, and 17.

In certain embodiments, the present invention contemplates makingfunctional variants by modifying the structure of an ActRIIB polypeptidefor such purposes as enhancing therapeutic efficacy, or stability (e.g.,ex vivo shelf life and resistance to proteolytic degradation in vivo).Modified ActRIIB polypeptides can also be produced, for instance, byamino acid substitution, deletion, or addition. For instance, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (e.g., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Whether a change inthe amino acid sequence of an ActRIIB polypeptide results in afunctional homolog can be readily determined by assessing the ability ofthe variant ActRIIB polypeptide to produce a response in cells in afashion similar to the wild-type ActRIIB polypeptide, or to bind to oneor more ligands, such as activin (e.g., activin A, activin B, activin C,and activin E), Nodal, GDF3, GDF-11, or myostatin in a fashion similarto wild type.

In certain embodiments, the present invention contemplates specificmutations of the ActRIIB polypeptides so as to alter the glycosylationof the polypeptide. Exemplary glycosylation sites in ActRIIBpolypeptides are illustrated in SEQ ID NO: 2. Such mutations may beselected so as to introduce or eliminate one or more glycosylationsites, such as 0-linked or N-linked glycosylation sites.Asparagine-linked glycosylation recognition sites generally comprise atripeptide sequence, asparagine-X-threonine (where “X” is any aminoacid) which is specifically recognized by appropriate cellularglycosylation enzymes. The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the wild-type ActRIIB polypeptide (for O-linkedglycosylation sites). A variety of amino acid substitutions or deletionsat one or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. Another means of increasing the number of carbohydratemoieties on an ActRIIB polypeptide is by chemical or enzymatic couplingof glycosides to the ActRIIB polypeptide. Depending on the coupling modeused, the sugar(s) may be attached to (a) arginine and histidine; (b)free carboxyl groups; (c) free sulfhydryl groups such as those ofcysteine; (d) free hydroxyl groups such as those of serine, threonine,or hydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Thesemethods are described in WO 87/05330 published Sep. 11, 1987, and inAplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306,incorporated by reference herein. Removal of one or more carbohydratemoieties present on an ActRIIB polypeptide may be accomplishedchemically and/or enzymatically. Chemical deglycosylation may involve,for example, exposure of the ActRIIB polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on ActRIIB polypeptides can be achieved by the use of a varietyof endo- and exo-glycosidases as described by Thotakura et al. (1987)Meth. Enzymol. 138:350. The sequence of an ActRIIB polypeptide may beadjusted, as appropriate, depending on the type of expression systemused, as mammalian, yeast, insect and plant cells may all introducediffering glycosylation patterns that can be affected by the amino acidsequence of the peptide. In general, ActRIIB proteins for use in humanswill be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines are expected to be useful as well.

This disclosure further contemplates a method of generating variants,particularly sets of combinatorial variants of an ActRIIB polypeptide,including, optionally, truncation variants; pools of combinatorialmutants are especially useful for identifying functional variantsequences. The purpose of screening such combinatorial libraries may beto generate, for example, ActRIIB polypeptide variants which havealtered properties, such as altered pharmacokinetics, or altered ligandbinding. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, an ActRIIBpolypeptide variant may be screened for ability to bind to an ActRIIBpolypeptide, to prevent binding of an ActRIIB ligand to an ActRIIBpolypeptide.

The activity of an ActRIIB polypeptide or its variants may also betested in a cell-based or in vivo assay. For example, the effect of anActRIIB polypeptide variant on the expression of genes involved inadipocyte differentiation or function may be assessed (e.g., UCP-1).This may, as needed, be performed in the presence of one or morerecombinant ActRIIB ligand protein (e.g., GDF8), and cells may betransfected so as to produce an ActRIIB polypeptide and/or variantsthereof, and optionally, an ActRIIB ligand. Likewise, an ActRIIBpolypeptide may be administered to a mouse or other animal, and one ormore properties of adipocytes, such as brown adipocyte thermogenesis maybe assessed. Similarly, the activity of an ActRIIB polypeptide or itsvariants may be tested in fat cells, muscle cells, bone cells, andneuronal cells for any effect on growth of these cells, for example, bythe assays as described below. Such assays are well known and routine inthe art. A SMAD-responsive reporter gene may be used in such cell linesto monitor effects on downstream signaling.

Combinatorially-derived variants can be generated which have a selectivepotency relative to a naturally occurring ActRIIB polypeptide. Suchvariant proteins, when expressed from recombinant DNA constructs, can beused in gene therapy protocols. Likewise, mutagenesis can give rise tovariants which have intracellular half-lives dramatically different thanthe corresponding a wild-type ActRIIB polypeptide. For example, thealtered protein can be rendered either more stable or less stable toproteolytic degradation or other processes which result in destructionof, or otherwise inactivation of a native ActRIIB polypeptide. Suchvariants, and the genes which encode them, can be utilized to alterActRIIB polypeptide levels by modulating the half-life of the ActRIIBpolypeptides. For instance, a short half-life can give rise to moretransient biological effects and, when part of an inducible expressionsystem, can allow tighter control of recombinant ActRIIB polypeptidelevels within the cell.

In certain embodiments, the ActRIIB polypeptides of the invention mayfurther comprise post-translational modifications in addition to anythat are naturally present in the ActRIIB polypeptides. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, the modified ActRIIB polypeptides may containnon-amino acid elements, such as polyethylene glycols, lipids, poly- ormono-saccharide, and phosphates. Effects of such non-amino acid elementson the functionality of a ActRIIB polypeptide may be tested as describedherein for other ActRIIB polypeptide variants. When an ActRIIBpolypeptide is produced in cells by cleaving a nascent form of theActRIIB polypeptide, post-translational processing may also be importantfor correct folding and/or function of the protein. Different cells(such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specificcellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the ActRIIB polypeptides.

In certain aspects, functional variants or modified forms of the ActRIIBpolypeptides include fusion proteins having at least a portion of theActRIIB polypeptides and one or more fusion domains. Well known examplesof such fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (e.g., an Fc),maltose binding protein (MBP), or human serum albumin. A fusion domainmay be selected so as to confer a desired property. For example, somefusion domains are particularly useful for isolation of the fusionproteins by affinity chromatography. For the purpose of affinitypurification, relevant matrices for affinity chromatography, such asglutathione-, amylase-, and nickel- or cobalt-conjugated resins areused. Many of such matrices are available in “kit” form, such as thePharmacia GST purification system and the QIAexpress™ system (Qiagen)useful with (HIS₆) fusion partners. As another example, a fusion domainmay be selected so as to facilitate detection of the ActRIIBpolypeptides. Examples of such detection domains include the variousfluorescent proteins (e.g., GFP) as well as “epitope tags,” which areusually short peptide sequences for which a specific antibody isavailable. Well known epitope tags for which specific monoclonalantibodies are readily available include FLAG, influenza virushaemagluttinin (HA), and c-myc tags. In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation. In certain preferred embodiments, an ActRIIBpolypeptide is fused with a domain that stabilizes the ActRIIBpolypeptide in vivo (a “stabilizer” domain). By “stabilizing” is meantanything that increases serum half life, regardless of whether this isbecause of decreased destruction, decreased clearance by the kidney, orother pharmacokinetic effect. Fusions with the Fc portion of animmunoglobulin are known to confer desirable pharmacokinetic propertieson a wide range of proteins. Likewise, fusions to human serum albumincan confer desirable properties. Other types of fusion domains that maybe selected include multimerizing (e.g., dimerizing, tetramerizing)domains and functional domains (that confer an additional biologicalfunction, such as further stimulation of muscle growth).

As a specific example, the present invention provides a fusion proteinas a GDF8 antagonist which comprises an extracellular (e.g.,GDF8-binding) domain fused to an Fc domain (e.g., SEQ ID NO: 13).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK

Optionally, the Fc domain has one or more mutations at residues such asAsp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asp-265 mutation) hasreduced ability of binding to the Fcγ receptor relative to a wildtype Fcdomain. In other cases, the mutant Fc domain having one or more of thesemutations (e.g., Asn-434 mutation) has increased ability of binding tothe MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fcdomain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ActRIIB polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to an ActRIIB polypeptide. The ActRIIBpolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

In certain embodiments, the ActRIIB polypeptides of the presentinvention contain one or more modifications that are capable ofstabilizing the ActRIIB polypeptides. For example, such modificationsenhance the in vitro half life of the ActRIIB polypeptides, enhancecirculatory half life of the ActRIIB polypeptides or reducingproteolytic degradation of the ActRIIB polypeptides. Such stabilizingmodifications include, but are not limited to, fusion proteins(including, for example, fusion proteins comprising an ActRIIBpolypeptide and a stabilizer domain), modifications of a glycosylationsite (including, for example, addition of a glycosylation site to anActRIIB polypeptide), and modifications of carbohydrate moiety(including, for example, removal of carbohydrate moieties from anActRIIB polypeptide). In the case of fusion proteins, an ActRIIBpolypeptide is fused to a stabilizer domain such as an IgG molecule(e.g., an Fc domain). As used herein, the term “stabilizer domain” notonly refers to a fusion domain (e.g., Fc) as in the case of fusionproteins, but also includes nonproteinaceous modifications such as acarbohydrate moiety, or nonproteinaceous polymer, such as polyethyleneglycol.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the ActRIIB polypeptides, which are isolatedfrom, or otherwise substantially free of, other proteins.

In certain embodiments, ActRIIB polypeptides (unmodified or modified) ofthe invention can be produced by a variety of art-known techniques. Forexample, such ActRIIB polypeptides can be synthesized using standardprotein chemistry techniques such as those described in Bodansky, M.Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) andGrant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman andCompany, New York (1992). In addition, automated peptide synthesizersare commercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, the ActRIIB polypeptides,fragments or variants thereof may be recombinantly produced usingvarious expression systems (e.g., E. coli, Chinese Hamster Ovary cells,COS cells, baculovirus) as is well known in the art (also see below). Ina further embodiment, the modified or unmodified ActRIIB polypeptidesmay be produced by digestion of naturally occurring or recombinantlyproduced full-length ActRIIB polypeptides by using, for example, aprotease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or pairedbasic amino acid converting enzyme (PACE). Computer analysis (using acommercially available software, e.g., MacVector, Omega, PCGene,Molecular Simulation, Inc.) can be used to identify proteolytic cleavagesites. Alternatively, such ActRIIB polypeptides may be produced fromnaturally occurring or recombinantly produced full-length ActRIIBpolypeptides such as standard techniques known in the art, such as bychemical cleavage (e.g., cyanogen bromide, hydroxylamine).

3. Nucleic Acids Encoding ActRIIB Polypeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the ActRIIB polypeptides (e.g., solubleActRIIB polypeptides), including any of the variants disclosed herein.For example, the following sequence encodes a naturally occurring humanActRIIB precursor polypeptide (SEQ ID NO: 4) (nucleotides 5-1543 ofNM_001106, 1539 bp):

atgacggcgccctgggtggccctcgccctcctctggggatcgctgtggcccggctctgggcgtggggaggctgagacacgggagtgcatctactacaacgccaactgggagctggagcgcaccaaccagagcggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgaaggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagcccccaccctgctcacggtgctggcctactcactgctgcccatcgggggcctttccctcatcgtcctgctggccttttggatgtaccggcatcgcaagcccccctacggtcatgtggacatccatgaggaccctgggcctccaccaccatcccctctggtgggcctgaagccactgcagctgctggagatcaaggctcgggggcgctttggctgtgtctggaaggcccagctcatgaatgactttgtagctgtcaagatcttcccactccaggacaagcagtcgtggcagagtgaacgggagatcttcagcacacctggcatgaagcacgagaacctgctacagttcattgctgccgagaagcgaggctccaacctcgaagtagagctgtggctcatcacggccttccatgacaagggctccctcacggattacctcaaggggaacatcatcacatggaacgaactgtgtcatgtagcagagacgatgtcacgaggcctctcatacctgcatgaggatgtgccctggtgccgtggcgagggccacaagccgtctattgcccacagggactttaaaagtaagaatgtattgctgaagagcgacctcacagccgtgctggctgactttggcttggctgttcgatttgagccagggaaacctccaggggacacccacggacaggtaggcacgagacggtacatggctcctgaggtgctcgagggagccatcaacttccagagagatgccttcctgcgcattgacatgtatgccatggggttggtgctgtgggagcttgtgtctcgctgcaaggctgcagacggacccgtggatgagtacatgctgccctttgaggaagagattggccagcacccttcgttggaggagctgcaggaggtggtggtgcacaagaagatgaggcccaccattaaagatcactggttgaaacacccgggcctggcccagctttgtgtgaccatcgaggagtgctgggaccatgatgcagaggctcgcttgtccgcgggctgtgtggaggagcgggtgtccctgattcggaggtcggtcaacggcactacctcggactgtctcgtttccctggtgacctctgtcaccaatgtggacctgcccc ctaaagagtcaagcatctaa

The following sequence encodes a human soluble (extracellular) ActRIIBpolypeptide (SEQ ID NO: 3) (348 bp).

tctgggcgtggggaggctgagacacgggagtgcatctactacaacgccaactgggagctggagcgcaccaaccagagcggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgaaggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagccc ccacc

The subject nucleic acids may be single-stranded or double stranded.Such nucleic acids may be DNA or RNA molecules. These nucleic acids aremay be used, for example, in methods for making ActRIIB polypeptides oras direct therapeutic agents (e.g., in a gene therapy approach).

In certain aspects, the subject nucleic acids encoding ActRIIBpolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NO: 3. Variant nucleotide sequences include sequencesthat differ by one or more nucleotide substitutions, additions ordeletions, such as allelic variants; and will, therefore, include codingsequences that differ from the nucleotide sequence of the codingsequence designated in SEQ ID NO: 4.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NO: 3. One of ordinary skill in the artwill appreciate that nucleic acid sequences complementary to SEQ ID NO:3, and variants of SEQ ID NO: 3 are also within the scope of thisinvention. In further embodiments, the nucleic acid sequences of theinvention can be isolated, recombinant, and/or fused with a heterologousnucleotide sequence, or in a DNA library. For example, the inventionprovides isolated or recombinant nucleic acid sequences that are atleast 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO:10 or 15.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NO: 3, complement sequenceof SEQ ID NO: 3, or fragments thereof. As discussed above, one ofordinary skill in the art will understand readily that appropriatestringency conditions which promote DNA hybridization can be varied. Oneof ordinary skill in the art will understand readily that appropriatestringency conditions which promote DNA hybridization can be varied. Forexample, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the invention provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NO: 3 due to degeneracy in the genetic code are also withinthe scope of the invention. For example, a number of amino acids aredesignated by more than one triplet. Codons that specify the same aminoacid, or synonyms (for example, CAU and CAC are synonyms for histidine)may result in “silent” mutations which do not affect the amino acidsequence of the protein. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject proteins will exist among mammalian cells. One skilled in theart will appreciate that these variations in one or more nucleotides (upto about 3-5% of the nucleotides) of the nucleic acids encoding aparticular protein may exist among individuals of a given species due tonatural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of thisinvention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the invention, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding an ActRIIB polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the ActRIIB polypeptide. Accordingly,the term regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding an ActRIIB polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant ActRIIB polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 1989) Chapters 16 and 17. In some instances, it may bedesirable to express the recombinant polypeptides by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ActRIIB polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject ActRIIB polypeptides in cells propagated in culture,e.g., to produce proteins, including fusion proteins or variantproteins, for purification.

This invention also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 3, 4, 10,or 15) for one or more of the subject ActRIIB polypeptide. The host cellmay be any prokaryotic or eukaryotic cell. For example, an ActRIIBpolypeptide of the invention may be expressed in bacterial cells such asE. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells. Other suitable host cells are known to thoseskilled in the art.

Accordingly, the present invention further pertains to methods ofproducing the subject ActRIIB polypeptides. For example, a host celltransfected with an expression vector encoding an ActRIIB polypeptidecan be cultured under appropriate conditions to allow expression of theActRIIB polypeptide to occur. The ActRIIB polypeptide may be secretedand isolated from a mixture of cells and medium containing the ActRIIBpolypeptide. Alternatively, the ActRIIB polypeptide may be retainedcytoplasmically or in a membrane fraction and the cells harvested, lysedand the protein isolated. A cell culture includes host cells, media andother byproducts. Suitable media for cell culture are well known in theart. The subject ActRIIB polypeptides can be isolated from cell culturemedium, host cells, or both, using techniques known in the art forpurifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for particularepitopes of the ActRIIB polypeptides. In a preferred embodiment, theActRIIB polypeptide is a fusion protein containing a domain whichfacilitates its purification.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ActRIIBpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ActRIIB polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Antibodies and Other Antagonists

Another aspect of the invention pertains to antibodies and otherantagonists, including proteins that bind to the targets disclosedherein and nucleic acids that inhibit expression of targets disclosedherein. An antibody that is specifically reactive with an ActRIIBpolypeptide (e.g., a soluble ActRIIB polypeptide) and which bindscompetitively with the ActRIIB polypeptide may be used as an antagonistof ActRIIB polypeptide activities. For example, by using immunogensderived from an ActRIIB polypeptide, anti-protein/anti-peptide antiseraor monoclonal antibodies can be made by standard protocols (see, forexample, Antibodies: A Laboratory Manual ed. by Harlow and Lane (ColdSpring Harbor Press: 1988)). A mammal, such as a mouse, a hamster orrabbit can be immunized with an immunogenic form of the ActRIIBpolypeptide or ligand, an antigenic fragment which is capable ofeliciting an antibody response, or a fusion protein. Techniques forconferring immunogenicity on a protein or peptide include conjugation tocarriers or other techniques well known in the art. An immunogenicportion of an ActRIIB polypeptide or ligand can be administered in thepresence of adjuvant. The progress of immunization can be monitored bydetection of antibody titers in plasma or serum. Standard ELISA or otherimmunoassays can be used with the immunogen as antigen to assess thelevels of antibodies.

Following immunization of an animal with an antigenic preparation of anActRIIB polypeptide or ligand, antisera can be obtained and, if desired,polyclonal antibodies can be isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, 1975, Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., 1983, Immunology Today, 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with an ActRIIBpolypeptide and monoclonal antibodies isolated from a culture comprisingsuch hybridoma cells.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with a subject ActRIIBpolypeptide or ligand. Antibodies can be fragmented using conventionaltechniques and the fragments screened for utility in the same manner asdescribed above for whole antibodies. For example, F(ab)₂ fragments canbe generated by treating antibody with pepsin. The resulting F(ab)₂fragment can be treated to reduce disulfide bridges to produce Fabfragments. The antibody of the present invention is further intended toinclude bispecific, single-chain, and chimeric and humanized moleculeshaving affinity for an ActRIIB polypeptide conferred by at least one CDRregion of the antibody. In preferred embodiments, the antibody furthercomprises a label attached thereto and able to be detected (e.g., thelabel can be a radioisotope, fluorescent compound, enzyme or enzymeco-factor).

In certain preferred embodiments, an antibody of the invention is amonoclonal antibody, and in certain embodiments, the invention makesavailable methods for generating novel antibodies. For example, a methodfor generating a monoclonal antibody that binds specifically to anActRIIB polypeptide or ligand may comprise administering to a mouse anamount of an immunogenic composition comprising the ActRIIB polypeptideor ligand effective to stimulate a detectable immune response, obtainingantibody-producing cells (e.g., cells from the spleen) from the mouseand fusing the antibody-producing cells with myeloma cells to obtainantibody-producing hybridomas, and testing the antibody-producinghybridomas to identify a hybridoma that produces a monoclonal antibodythat binds specifically to the ActRIIB polypeptide or ligand. Onceobtained, a hybridoma can be propagated in a cell culture, optionally inculture conditions where the hybridoma-derived cells produce themonoclonal antibody that binds specifically to the ActRIIB polypeptideor ligand. The monoclonal antibody may be purified from the cellculture.

The adjective “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g., an ActRIIB polypeptide) and other antigens that are notof interest that the antibody is useful for, at minimum, detecting thepresence of the antigen of interest in a particular type of biologicalsample. In certain methods employing the antibody, such as therapeuticapplications, a higher degree of specificity in binding may bedesirable. Monoclonal antibodies generally have a greater tendency (ascompared to polyclonal antibodies) to discriminate effectively betweenthe desired antigens and cross-reacting polypeptides. One characteristicthat influences the specificity of an antibody:antigen interaction isthe affinity of the antibody for the antigen. Although the desiredspecificity may be reached with a range of different affinities,generally preferred antibodies will have an affinity (a dissociationconstant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or less.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore binding assay, Bia-core AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

In certain aspects, the disclosure provides antibodies that bind to asoluble ActRIIB polypeptide or ligand. Such antibodies may be generatedmuch as described above, using a soluble ActRIIB polypeptide or ligandor fragment thereof as an antigen. Antibodies of this type can be used,e.g., to detect ActRIIB polypeptides in biological samples and/or tomonitor soluble ActRIIB polypeptide levels in an individual. In certaincases, an antibody that specifically binds to a soluble ActRIIBpolypeptide or ligand can be used to modulate activity of an ActRIIBpolypeptide and/or an ActRIIB ligand, thereby increasing thermogenicadipocytes.

Certain ligands, such as myostatin and GDF3 may be inhibited by using apolypeptide comprising a binding portion of the respective propeptide,or a variant thereof. Such propeptides may be prepared as fusionproteins, including Fc fusion proteins. Examples of suitable propeptidesare disclosed in published patent applications WO 02/085306 and WO06/002387.

Additionally, other binding proteins, such as the so-called “traps”(e.g., follistatin, FLRG, FSTL, Cerberus and Coco), soluble type Ireceptors, e.g., ALK-7 may be used. Examples of such polypeptides may befound in published patent applications WO 05/115439, WO 08/109779, WO08/067480, WO 07/109686, WO 05/100563, and WO 05/025601.

Nucleic acids, such as antisense or RNAi probes (which may include bothnaturally and non-naturally occurring nucleotides) may be used toinhibit expression of ActRIIB or any of the ligands discussed herein.

5. Screening Assays

In certain aspects, the present invention relates to the use of thesubject ActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) toidentify compounds (agents) which are agonist or antagonists of theActRIIB polypeptides. Compounds identified through this screening can betested in tissues such as fat, muscle, bone, cartilage, and/or neurons,to assess their ability to modulate tissue growth in vitro. Optionally,these compounds can further be tested in animal models to assess theirability to modulate tissue growth in vivo.

There are numerous approaches to screening for therapeutic agents formodulating tissue growth by targeting the ActRIIB polypeptides. Incertain embodiments, high-throughput screening of compounds can becarried out to identify agents that perturb ActRIIB-mediated effects ongrowth of fat, muscle, bone, cartilage, and/or neurons. In certainembodiments, the assay is carried out to screen and identify compoundsthat specifically inhibit or reduce binding of an ActRIIB polypeptide toits binding partner, such as an ActRIIB ligand (e.g., activin, GDF3,Nodal, GDF8, or GDF11). Alternatively, the assay can be used to identifycompounds that enhance binding of an ActRIIB polypeptide to its bindingprotein such as an ActRIIB ligand. In a further embodiment, thecompounds can be identified by their ability to interact with an ActRIIBpolypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatable crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between an ActRIIBpolypeptide and its binding protein (e.g., an ActRIIB ligand).

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified ActRIIB polypeptide which is ordinarily capable of binding toan ActRIIB ligand, as appropriate for the intention of the assay. To themixture of the compound and ActRIIB polypeptide is then added acomposition containing an ActRIIB ligand. Detection and quantificationof ActRIIB/ActRIIB ligand complexes provides a means for determining thecompound's efficacy at inhibiting (or potentiating) complex formationbetween the ActRIIB polypeptide and its binding protein. The efficacy ofthe compound can be assessed by generating dose response curves fromdata obtained using various concentrations of the test compound.Moreover, a control assay can also be performed to provide a baselinefor comparison. For example, in a control assay, isolated and purifiedActRIIB ligand is added to a composition containing the ActRIIBpolypeptide, and the formation of ActRIIB/ActRIIB ligand complex isquantitated in the absence of the test compound. It will be understoodthat, in general, the order in which the reactants may be admixed can bevaried, and can be admixed simultaneously. Moreover, in place ofpurified proteins, cellular extracts and lysates may be used to render asuitable cell-free assay system.

Complex formation between the ActRIIB polypeptide and its bindingprotein may be detected by a variety of techniques. For instance,modulation of the formation of complexes can be quantitated using, forexample, detectably labeled proteins such as radiolabeled (e.g., ³²P,³⁵S, ¹⁴C or ³H), fluorescently labeled (e.g., FITC), or enzymaticallylabeled ActRIIB polypeptide or its binding protein, by immunoassay, orby chromatographic detection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between an ActRIIB polypeptide and its bindingprotein. Further, other modes of detection, such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between an ActRIIB polypeptideand its binding protein. See for example, U.S. Pat. No. 5,283,317;Zervos et al., 1993, Cell 72:223-232; Madura et al., 1993, J Biol Chem268:12046-12054; Bartel et al., 1993, Biotechniques 14:920-924; andIwabuchi et al., 1993, Oncogene 8:1693-1696). In a specific embodiment,the present invention contemplates the use of reverse two hybrid systemsto identify compounds (e.g., small molecules or peptides) thatdissociate interactions between an ActRIIB polypeptide and its bindingprotein. See for example, Vidal and Legrain, 1999, Nucleic Acids Res27:919-29; Vidal and Legrain, 1999, Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, the subject compounds are identified by theirability to interact with an ActRIIB polypeptide of the invention. Theinteraction between the compound and the ActRIIB polypeptide may becovalent or non-covalent. For example, such interaction can beidentified at the protein level using in vitro biochemical methods,including photo-crosslinking, radiolabeled ligand binding, and affinitychromatography (Jakoby W B et al., 1974, Methods in Enzymology 46: 1).In certain cases, the compounds may be screened in a mechanism basedassay, such as an assay to detect compounds which bind to an ActRIIBpolypeptide. This may include a solid phase or fluid phase bindingevent. Alternatively, the gene encoding an ActRIIB polypeptide can betransfected with a reporter system (e.g., (3-galactosidase, luciferase,or green fluorescent protein) into a cell and screened against thelibrary preferably by a high throughput screening or with individualmembers of the library. Other mechanism based binding assays may beused, for example, binding assays which detect changes in free energy.Binding assays can be performed with the target fixed to a well, bead orchip or captured by an immobilized antibody or resolved by capillaryelectrophoresis. The bound compounds may be detected usually usingcolorimetric or fluorescence or surface plasmon resonance.

In certain aspects, the present invention provides methods and agentsfor controlling weight gain and obesity. At the cellular level,adipocyte proliferation and differentiation is critical in thedevelopment of obesity, which leads to the generation of additional fatcells (adipocytes). Therefore, any compound identified can be tested inwhole cells or tissues, in vitro or in vivo, to confirm their ability tomodulate adipogenesis by measuring adipocyte proliferation ordifferentiation. Various methods known in the art can be utilized forthis purpose. For example, the effect of an ActRIIB polypeptide (e.g., asoluble ActRIIB polypeptide) or test compounds on adipogenesis can bedetermined by measuring differentiation of 3T3-L1 preadipocytes tomature adipocytes in cell based assays, such as, by observing theaccumulation of triacylglycerol in Oil Red O staining vesicles and bythe appearance of certain adipocyte markers such as FABP (aP2/422) andPPARγ2. See, for example, Reusch et al., 2000, Mol Cell Biol.20:1008-20; Deng et al., 2000, Endocrinology. 141:2370-6; Bell et al.,2000, Obes Res. 8:249-54. Another example of cell-based assays includesanalyzing the role of ActRIIB polypeptides and test compounds inproliferation of adipocytes or adipocyte precursor cells (e.g., 3T3-L1cells), such as, by monitoring bromodeoxyuridine (BrdU)-positive cells.See, for example, Pico et al., 1998, Mol Cell Biochem. 189:1-7; Masunoet al., 2003, Toxicol Sci. 75:314-20.

It is understood that the screening assays of the present inventionapply to not only the subject ActRIIB polypeptides and variants of theActRIIB polypeptides, but also any test compounds including agonists andantagonist of the ActRIIB polypeptides or ActRIIB signaling. Further,these screening assays are useful for drug target verification andquality control purposes.

6. Exemplary Therapeutic Uses

In certain embodiments, compositions (e.g., ActRIIB polypeptides) of thepresent invention can be used for treating or preventing a disease orcondition that is associated with abnormal activity of an ActRIIBpolypeptide and/or an ActRIIB ligand (e.g., activin or GDF8). In certainembodiments, the present invention provides methods of treating orpreventing an individual in need thereof through administering to theindividual a therapeutically effective amount of an ActRIIB polypeptideas described above. These methods are particularly aimed at therapeuticand prophylactic treatments of animals, and more particularly, humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes prophylaxis of the named condition or amelioration orelimination of the condition once it has been established.

As demonstrated herein, ActRIIB-Fc promotes the expression of UCP1, aprotein that mediates an uncoupling in mitochondria, leading tometabolically active, or thermogenic, adipose tissue. Accordingly,compositions disclosed herein may be used to treat a variety ofdisorders, such as a deficiency in brown adipose tissue or brownadipocytes, metabolic syndrome (also known as syndrome X), diabetes,hyperlipidemia, hypercholesterolemia, overeating and bulimia,hypertension, arteriosclerosis (coronary artery disease or coronaryheart disease), myocardial infarction, congestive heart failure,cerebral infarction, cerebral thrombosis, respiratory disorders (such asPickwickian syndrome), cancers of the colon, prostate, breast,endometrium, and kidney, growth hormone-deficient subjects, normalvariant short stature, Turner's syndrome, and other pathologicalconditions showing reduced metabolic activity or a decrease in restingenergy expenditure as a percentage of total fat-free mass, e.g.,children with acute lymphoblastic leukemia.

In certain embodiments, compositions (e.g., soluble ActRIIBpolypeptides) of the invention are used to promote formation and/oractivity of thermogenic adipocytes. As discussed above, thermogenicdiscrete brown-adipose tissue and brown adiopocytes within white adiposetissue contain large numbers of mitochondria expressing uncouplingprotein-1 (UCP). Individuals with high caloric intake and lacking brownadipocytes are unable to convert excess caloric intake to heat and aretherefore compelled to store unused biochemical energy, typically asenlarged white adipose tissue. Blocking or antagonizing function of oneor more ActRIIB ligands (e.g., GDF8) in vivo can effectively increasethermogenic activity of brown adipocytes in discrete depots or of brownadiopocytes distributed within white adipose tissue. This approach isconfirmed and supported by the data shown herein, whereby an ActRIIB-Fcprotein was shown to induce UCP1 expression in white fat, enhanceoverall body composition, and improve metabolic status in mice on ahigh-fat diet.

In certain embodiments, compositions (e.g., soluble ActRIIBpolypeptides) of the invention are used as part of a treatment formetabolic syndrome (also known as syndrome X and insulin resistancesyndrome), which is a combination of disorders and risk factors thatincrease the risk of developing cardiovascular disease and diabetesmellitus type II. Most patients are older, obese, sedentary, and havesome degree of insulin resistance. Central (abdominal or visceral)adiposity is a significant feature of the syndrome.

In related embodiments, soluble ActRIIB polypeptides and othercompositions of the invention can be used as part of a treatment fordiabetes mellitus type II (also known as non-insulin-dependent diabetesmellitus or adult-onset diabetes), which is characterized by elevatedblood glucose in the context of insulin resistance and relative insulindeficiency. Complex and multifactorial metabolic changes in diabetesoften lead to damage and functional impairment of many organs, mostimportantly the cardiovascular system. Diabetes mellitus type II isoften associated with obesity (abdominal or visceral adiposity),hypertension, elevated cholesterol, and metabolic syndrome. Importantrisk factors for diabetes mellitus type II include aging, high-fatdiets, and a sedentary lifestyle.

In other related embodiments, soluble ActRIIB polypeptides and othercompositions of the invention can be used as part of a treatment foratherosclerosis, a chronic inflammatory condition in which artery wallsthicken due to the accumulation of fatty deposits, often referred to asplaques. Risk factors for atherosclerosis include aging, diabetesmellitus, dyslipoproteinemia, obesity (abdominal or visceral adiposity),and a sedentary lifestyle.

Soluble ActRIIB polypeptides can also be used for lipodystrophicdisorders, which tend to be associated with metabolic syndrome. Severeinsulin resistance can result from both genetic and acquired forms oflipodystrophy, including in the latter case human immunodeficiency virus(HIV)-related lipodystrophy in patients treated with antiretroviraltherapy.

The subject ActRIIB polypeptides may further be used as a therapeuticagent for slowing or preventing the development of obesity. Thisapproach is confirmed and supported by the data shown herein, whereby anActRIIB-Fc protein was shown to improve metabolic status in mice on ahigh-fat diet.

In other embodiments, the present invention provides compositions andmethods for regulating body fat content in an animal and for treating orpreventing conditions related thereto, and particularly,health-compromising conditions related thereto. According to the presentinvention, to regulate (control) body weight can refer to reducing orincreasing body weight, reducing or increasing the rate of weight gain,or increasing or reducing the rate of weight loss, and also includesactively maintaining, or not significantly changing body weight (e.g.,against external or internal influences which may otherwise increase ordecrease body weight). One embodiment of the present invention relatesto regulating body weight by administering to an animal (e.g., a human)in need thereof an ActRIIB polypeptide.

In one specific embodiment, the present invention relates to methods andcompounds for reducing body weight and/or reducing weight gain in ananimal, and more particularly, for treating or ameliorating obesity inpatients at risk for or suffering from obesity. In another specificembodiment, the present invention is directed to methods and compoundsfor treating an animal that is unable to gain or retain weight (e.g., ananimal with a wasting syndrome). Such methods are effective to increasebody weight and/or mass, or to reduce weight and/or mass loss, or toimprove conditions associated with or caused by undesirably low (e.g.,unhealthy) body weight and/or mass.

As demonstrated in WO 2006/012627 and WO 2008/097541, compoundsdisclosed herein stimulate muscle growth. Accordingly, these compoundsmay be particularly useful in diseases or conditions with overlappingmuscle and metabolic dysfunction.

In certain embodiments, compositions (e.g., soluble ActRIIBpolypeptides) of the invention are used as part of a treatment for amuscular dystrophy. The term “muscular dystrophy” refers to a group ofdegenerative muscle diseases characterized by gradual weakening anddeterioration of skeletal muscles and sometimes the heart andrespiratory muscles. Muscular dystrophies are genetic disorderscharacterized by progressive muscle wasting and weakness that begin withmicroscopic changes in the muscle. As muscles degenerate over time, theperson's muscle strength declines. Moreover, declining muscle mass anddiminishing physical activity contribute to an imbalance between caloricintake and energy expenditure, leading to unhealthy storage of excessenergy as white adipose tissue. Exemplary muscular dystrophies that canbe treated with a regimen including the subject ActRIIB polypeptidesinclude: Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy(BMD), Emery-Dreifuss Muscular Dystrophy (EDMD), Limb-Girdle MuscularDystrophy (LGMD), Facioscapulohumeral Muscular Dystrophy (FSH or FSHD)(also known as Landouzy-Dejerine), Myotonic Dystrophy (MMD) (also knownas Steinert's Disease), Oculopharyngeal Muscular Dystrophy (OPMD),Distal Muscular Dystrophy (DD), Congenital Muscular Dystrophy (CMD).

Duchenne Muscular Dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. BeckerMuscular Dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is broken. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

BMD results from different mutations in the dystrophin gene. BMDpatients have some dystrophin, but it is either insufficient in quantityor poor in quality. Having some dystrophin protects the muscles of thosewith BMD from degenerating as badly or as quickly as those of peoplewith DMD.

For example, recent researches demonstrate that blocking or eliminatingfunction of GDF8 (an ActRIIB ligand) in vivo can effectively treat atleast certain symptoms in DMD and BMD patients. Thus, the subjectActRIIB polypeptides may act as GDF8 inhibitors (antagonists), andconstitute an alternative means of blocking the functions of GDF8 and/orActRIIB in vivo in DMD and BMD patients. This approach is confirmed andsupported by the data shown herein, whereby an ActRIIB-Fc protein wasshown to increase muscle mass in a mouse model of muscular dystrophy.

Similarly, the subject ActRIIB polypeptides provide an effective meansto increase muscle mass in other disease conditions that are in need ofmuscle growth. For example, ALS, also called Lou Gehrig's disease (motorneuron disease) is a chronic, incurable, and unstoppable CNS disorderthat attacks the motor neurons, components of the CNS that connect thebrain to the skeletal muscles. In ALS, the motor neurons deteriorate andeventually die, and though a person's brain normally remains fullyfunctioning and alert, the command to move never reaches the muscles.Most people who get ALS are between 40 and 70 years old. The first motorneurons that weaken are those leading to the arms or legs. Those withALS may have trouble walking, they may drop things, fall, slur theirspeech, and laugh or cry uncontrollably. Eventually the muscles in thelimbs begin to atrophy from disuse. This muscle weakness will becomedebilitating and a person will need a wheel chair or become unable tofunction out of bed. Most ALS patients die from respiratory failure orfrom complications of ventilator assistance like pneumonia, 3-5 yearsfrom disease onset. This approach is confirmed and supported by the datashown herein, whereby an ActRIIB-Fc protein was shown to improve theappearance, muscle mass and lifespan of a mouse model of ALS.

ActRIIB polypeptide-induced increased muscle mass might also benefitthose suffering from muscle wasting diseases. Gonzalez-Cadavid et al.(supra) reported that that GDF8 expression correlates inversely withfat-free mass in humans and that increased expression of the GDF8 geneis associated with weight loss in men with AIDS wasting syndrome. Byinhibiting the function of GDF8 in AIDS patients, at least certainsymptoms of AIDS may be alleviated, if not completely eliminated, thussignificantly improving quality of life in AIDS patients.

Sarcopenia, the loss of muscle with aging is also often associated withmetabolic syndrome, diabetes, arteriosclerosis, dyslipidemia, and otherage-related metabolic conditions. ActRIIB polypeptide-induced musclemass might also benefit those suffering from sarcopenia.

7. Pharmaceutical Compositions

In certain embodiments, compounds (e.g., ActRIIB polypeptides) of thepresent invention are formulated with a pharmaceutically acceptablecarrier. For example, an ActRIIB polypeptide can be administered aloneor as a component of a pharmaceutical formulation (therapeuticcomposition). The subject compounds may be formulated for administrationin any convenient way for use in human or veterinary medicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition topically, systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this invention is, of course, in a pyrogen-free, physiologicallyacceptable form. Further, the composition may desirably be encapsulatedor injected in a viscous form for delivery to a target tissue site(e.g., bone, cartilage, muscle, fat or neurons), for example, a sitehaving a tissue damage. Topical administration may be suitable for woundhealing and tissue repair. Therapeutically useful agents other than theActRIIB polypeptides which may also optionally be included in thecomposition as described above, may alternatively or additionally, beadministered simultaneously or sequentially with the subject compounds(e.g., ActRIIB polypeptides) in the methods of the invention.

In certain embodiments, compositions of the present invention mayinclude a matrix capable of delivering one or more therapeutic compounds(e.g., ActRIIB polypeptides) to a target tissue site, providing astructure for the developing tissue and optimally capable of beingresorbed into the body. For example, the matrix may provide slow releaseof the ActRIIB polypeptides. Such matrices may be formed of materialspresently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Certain compositions disclosed herein may be administered topically,either to skin or to mucosal membranes. The topical formulations mayfurther include one or more of the wide variety of agents known to beeffective as skin or stratum corneum penetration enhancers. Examples ofthese are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and azone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to a subjectcompound of the invention (e.g., an ActRIIB polypeptide), excipients,such as animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a subject compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

In certain embodiments, pharmaceutical compositions suitable forparenteral administration may comprise one or more ActRIIB polypeptidesin combination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., ActRIIB polypeptides).The various factors will depend upon the disease to be treated.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of ActRIIB polypeptides or other compoundsdisclosed herein. Such therapy would achieve its therapeutic effect byintroduction of the ActRIIB polynucleotide sequences into cells ortissues having the disorders as listed above. Delivery of ActRIIBpolynucleotide sequences can be achieved using a recombinant expressionvector such as a chimeric virus or a colloidal dispersion system.Preferred for therapeutic delivery of ActRIIB polynucleotide sequencesis the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the ActRIIB polynucleotide. In one preferredembodiment, the vector is targeted to bone, cartilage, muscle or neuroncells/tissues.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for ActRIIB polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1. Generation of an ActRIIB-Fc Fusion Protein

Applicants constructed a soluble ActRIIB fusion protein that has theextracellular domain of human ActRIIB fused to a human or mouse Fcdomain with a minimal linker (three glycine amino acids) in between. Theconstructs are referred to as ActRIIB(20-134)-hFc andActRIIB(20-134)-mFc, respectively.

ActRIIB-hFc is shown below as purified from CHO cell lines (SEQ ID NO:5)

GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The ActRIIB(20-134)-hFc and ActRIIB(20-134)-mFc proteins were expressedin CHO cell lines. Three different leader sequences were considered:

(i) Honey bee mellitin (HBML): (SEQ ID NO: 7) MKFLVNVALVFMVVYISYIYA(ii) Tissue Plasminogen Activator (TPA): (SEQ ID NO: 8)MDAMKRGLCCVLLLCGAVFVSP (iii) Native: (SEQ ID NO: 9)MGAAAKLAFAVFLISCSSGA.

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence:

(SEQ ID NO: 17) MDAMKRGLCCVLLLCGAVFVSPGASGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence (SEQID NO:10):

A TGGATGCAAT GAAGAGAGGG CTCTGCTGTG TGCTGCTGCT GTGTGGAGCA GTCTTCGTTTCGCCCGGCGC CTCTGGGCGT GGGGAGGCTG AGACACGGGA GTGCATCTAC TACAACGCCAACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTGGAGCG CTGCGAAGGC GAGCAGGACAAGCGGCTGCA CTGCTACGCC TCCTGGCGCA ACAGCTCTGG CACCATCGAG CTCGTGAAGAAGGGCTGCTG GCTAGATGAC TTCAACTGCT ACGATAGGCA GGAGTGTGTG GCCACTGAGGAGAACCCCCA GGTGTACTTC TGCTGCTGTG AAGGCAACTT CTGCAACGAG CGCTTCACTCATTTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTACGAGCC ACCCCCGACA GCCCCCACCGGTGGTGGAAC TCACACATGC CCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAGTCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCACATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGGACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGTACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACAAGTGCAAGGT CTCCAACAAA GCCCTCCCAG TCCCCATCGA GAAAACCATC TCCAAAGCCAAAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCAAGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGGAGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACTCCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGGGGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGAGCCTCTCCCT GTCTCCGGGT AAATGA

N-terminal sequencing of the CHO-cell produced material revealed a majorsequence of -GRGEAE (SEQ ID NO: 11). Notably, other constructs reportedin the literature begin with an -SGR . . . sequence.

Purification could be achieved by a series of column chromatographysteps, including, for example, three or more of the following, in anyorder: protein A chromatography, Q sepharose chromatography,phenylsepharose chromatography, size exclusion chromatography, andcation exchange chromatography. The purification could be completed withviral filtration and buffer exchange.

ActRIIB-Fc fusion proteins were also expressed in HEK293 cells and COScells. Although material from all cell lines and reasonable cultureconditions provided protein with muscle-building activity in vivo,variability in potency was observed perhaps relating to cell lineselection and/or culture conditions.

Example 2: Generation of ActRIIB-Fc Mutants

Applicants generated a series of mutations in the extracellular domainof ActRIIB and produced these mutant proteins as soluble fusion proteinsbetween extracellular ActRIIB and an Fc domain. The backgroundActRIIB-Fc fusion has the sequence (Fc portion underlined)(SEQ IDNO:12):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K

Various mutations, including N- and C-terminal truncations, wereintroduced into the background ActRIIB-Fc protein. Based on the datapresented in Example 1, it is expected that these constructs, ifexpressed with a TPA leader, will lack the N-terminal serine.

Mutations were generated in ActRIIB extracellular domain by PCRmutagenesis. After PCR, fragments were purified through a Qiagen column,digested with SfoI and AgeI and gel purified. These fragments wereligated into expression vector pAID4 (see WO2006/012627) such that uponligation it created fusion chimera with human IgG1. Upon transformationinto E. coli DH5 alpha, colonies were picked and DNAs were isolated. Formurine constructs (mFc), a murine IgG2a was substituted for the humanIgG1. All mutants were sequence verified.

All of the mutants were produced in HEK293T cells by transienttransfection. In summary, in a 500 ml spinner, HEK293T cells were set upat 6×10⁵ cells/ml in Freestyle (Invitrogen) media in 250 ml volume andgrown overnight. Next day, these cells were treated with DNA:PEI (1:1)complex at 0.5 ug/ml final DNA concentration. After 4 hrs, 250 ml mediawas added and cells were grown for 7 days. Conditioned media washarvested by spinning down the cells and concentrated.

Mutants were purified using a variety of techniques, including, forexample, protein A column and eluted with low pH (3.0) glycine buffer.After neutralization, these were dialyzed against PBS.

Mutants were also produced in CHO cells by similar methodology.

Mutants were tested in binding assays and/or bioassays. In someinstances, assays were performed with conditioned medium rather thanpurified proteins. Variants are described, for example, in publishedpatent applications WO 06/012627 and WO 08/097541. Such variants may beused in the methods described herein.

Example 3: Effect of ActRIIB(20-134)-hFc on Thermogenic Properties ofWhite Adipose Tissue in Mice Fed a High-Fat Diet

Applicants investigated the effects of ActRIIB-Fc on brown adipocytesand other metabolic endpoints in male mice fed a high-fat diet.Ten-week-old C57BL/6 mice were weight-matched and treated withActRIIB(20-134)-hFc (n=10) or Tris-buffered-saline (TBS) vehicle (n=7)twice per week at 10 mg/kg, s.c., for 60 days. During this period, micehad unlimited access to a diet containing 58% fat instead of thestandard chow containing 4.5% fat. At study termination, epididymal fatpads were collected, and quantitative RT-PCR (reverse transcriptionpolymerase chain reaction) was used to measure levels of mRNA encodinguncoupling protein-1 (UCP1), a well-documented marker of thermogeniccapability in brown adipocytes, which are diffusely distributed withinwhite adipose depots (Cousin et al., 1992, J Cell Sci 103:931-942).

ActRIIB(20-134)-hFc treatment caused a constellation of noteworthymetabolic effects. In mice on the high-fat diet, ActRIIB(20-134)-hFcincreased UCP1 mRNA levels in epididymal fat nearly nine-fold comparedto vehicle (FIG. 1; P<0.05), a particularly impressive effect given thatC57BL/6 mice display severely blunted induction of UCP1 and brownadipocytes within key white fat depots compared to other mouse strains(Guerra et al., 1998, J Clin Invest 102:412-420; Xue et al., 2007, JLipid Res 48:41-51). ActRIIB(20-134)-hFc also produced a beneficial, 30%reduction (P<0.001) of serum free fatty acid concentrations.Importantly, upregulation of UCP1 was accompanied by beneficial effectsof ActRIIB(20-134)-hFc on body composition, as determined by nuclearmagnetic resonance (NMR) at baseline and Day 48. Under high-fat dietaryconditions, total fat mass in vehicle-treated controls tripled duringthis 48-day period, and ActRIIB(20-134)-hFc treatment cut this increaseby 40%. By Day 48, total fat mass was 26% of body weight inActRIIB(20-134)-hFc-treated mice vs. 39% in control mice, whereas leantissue mass was 64% of body weight in ActRIIB-Fc-treated mice vs. 55% incontrol mice. Thus, the net result was a healthier body compositionunder conditions of high-fat diet.

Example 4: Effect of Truncated Variant ActRIIB(25-131)-hFc onThermogenic Properties of White Adipose Tissue in Mice Fed a High-FatDiet

In the study described above (Example 3), Applicants also investigatedeffects of the truncated variant ActRIIB(25-131)-hFc on thermogenicproperties of white adipose tissue and other metabolic endpoints underhigh-fat dietary conditions.

Applicants generated a truncated fusion protein ActRIIB(25-131)-hFc(FIGS. 13-14), using the same leader and methodology as described abovewith respect to ActRIIB(20-134)-hFc. The mature protein purified afterexpression in CHO cells has the sequence shown below (SEQ ID NO: 6):

ETRECIYYNA NWELERTNQS GLERCEGEQD KRLHCYASWRNSSGTIELVK KGCWLDDFNC YDRQECVATE ENPQVYFCCCEGNFCNERFT HLPEAGGPEV TYEPPPTGGG THTCPPCPAPELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQDWLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLPPSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK

Ten-week-old C57BL/6 mice were treated with ActRIIB(25-131)-hFc, at 10mg/kg, s.c., or Tris-buffered-saline (TBS) vehicle twice per week for 60days. During this period, mice had unlimited access to a diet containing58% fat instead of the standard chow containing 4.5% fat. An additionalgroup of mice maintained on the standard chow diet were also treatedwith TBS vehicle and followed as a dietary control.

Under high-fat dietary conditions, ActRIIB(25-131)-hFc treatmenttriggered histological changes and a gene expression profile in whiteadipose tissue that were consistent with thermogenic capability. Asshown in FIG. 2, histological examination of epididymal white fatindicated that ActRIIB(25-131)-hFc reduced lipid droplet size and causedformation of clusters of multilocular adipocytes that are a hallmark ofbrown fat. Moreover, immunohistochemical analysis of this tissuerevealed widespread cytoplasmic induction of UCP1 in both multilocularand unilocular adipocytes as a result of ActRIIB(25-131)-hFc treatment(FIG. 2).

Accompanying these histological changes were significant changes in theexpression of key thermogenic and metabolic regulatory genes inepididymal white fat, as determined by quantitative RT-PCR. In mice onthe high-fat diet, ActRIIB(25-131)-hFc treatment increased UCP1 mRNAlevels more than 60-fold compared to vehicle (FIG. 3), a particularlyimpressive change since, as noted above, this strain of mouse displaysseverely blunted induction of UCP1 and brown adipocytes within key whitefat depots compared to other mouse strains. In addition,ActRIIB(25-131)-hFc treatment increased levels of mRNA encoding thesirtuin SIRT-1 (silent information regulator two, homolog 1) (FIG. 4),an energy-sensitive master regulator (deacetylase) that protects againstmetabolic damage induced by a high-fat diet (Pfluger et al., 2008, ProcNatl Acad Sci USA 105:9793-9798) and is implicated as an importantcontrol of fatty acid mobilization (Rodgers et al., 2008, FEBS Lett582:46-53). Significantly, ActRIIB(25-131)-hFc treatment also increasedlevels of mRNA encoding PGC-1α (peroxisome proliferator-activatedreceptor gamma coactivator-1a) (FIG. 5), a well-documented target ofSIRT-1 that, in turn, controls expression of many genes necessary formitochondrial biogenesis and thermogenic capability in brown adioposetissue (Uldry et al., 2006, Cell Metab, 3:333-341). Notably, forcedexpression of PGC-1α in white adipocytes has been shown to induce athermogenic program of gene expression, including UCP1, closelyresembling that in brown adipocytes (Hansen et al., 2006, Biochem J398:153-168). In the present study, ActRIIB(25-131)-hFc restored PGC-1αgene expression in white adipose tissue under high-fat dietaryconditions to levels indistinguishable from those in mice fed thestandard diet (FIG. 5).

Additional changes associated with treatment constitute a prominent linkbetween the altered expression profile in white adipose tissue andbeneficial hormonal and metabolic effects. Thus, in epididymal whitefat, ActRIIB(25-131)-hFc increased levels of mRNA encoding Foxo-1(forkhead box-containing, protein O subfamily-1) (FIG. 6), atranscription factor that is both a target of SIRT-1 and a key inducerof adiponectin expression (Qiao et al., 2006, J Biol Chem281:39915-39924). Adiponectin, a fat-derived hormone whose concentrationvaries inversely with fat mass/obesity, exerts importantinsulin-sensitizing actions in target tissues (Yamauchi et al., 2001,Nat Med 7:941-946; Maeda et al., 2002, Nat Med 8:731-737; Kadowaki etal., 2005, Endocr Rev 26:439-451). Consistent with Foxo-1 mRNAinduction, ActRIIB(25-131)-hFc treatment raised levels of adiponectinmRNA in epididymal white fat (FIG. 7) as well as circulatingconcentrations of adiponectin (FIG. 8). Importantly, these changes wereaccompanied in ActRIIB(25-131)-hFc-treated mice by robust decreases incirculating insulin (FIG. 9), triglycerides, free fatty acids,high-density lipoprotein (HDL), and low-density lipoprotein (LDL),leading to normalization of nearly all of these parameters. Finally, theaforementioned effects were accompanied by beneficial changes in bodycomposition, as determined by nuclear magnetic resonance (NMR) atbaseline and Day 48. Specifically, total fat mass in vehicle-treatedcontrols under high-fat dietary conditions tripled during this 48-dayperiod, and ActRIIB(25-131)-hFc treatment cut this increase by nearly40%. In summary, ActRIIB(25-131)-hFc treatment under high-fat dietaryconditions resulted in 1) histological changes and a gene expressionprofile in white adipose tissue that were consistent with thermogeniccapability, 2) beneficial changes in a wide range of hormonal andmetabolic parameters, and 3) improved body composition.

Example 5: Effect of ActRIIB(25-131)-mFc on Brown Fat Depots in Mice Feda High-Fat Diet

In another study, Applicants investigated effects of the truncatedvariant ActRIIB(25-131)-mFc on properties of intracapsular brown fatdepots under high-fat dietary conditions. Nine-week-old C57BL/6 micewere treated with ActRIIB(25-131)-mFc (n=20), at 10 mg/kg, s.c., orTris-buffered-saline (TBS) vehicle (n=10) twice per week for 60 days.Beginning 7 days before the start of dosing, mice had unlimited accessto a diet containing 58% fat instead of the standard chow containing4.5% fat. An additional group of mice (n=10) maintained on the standardchow diet were also treated with TBS vehicle and followed as a dietarycontrol.

Compared to the standard diet, the high-fat diet produced severalnoticeable changes in the interscapular depot of brown adipose tissue,and ActRIIB(25-131)-mFc treatment either completely or largely reversedeach of these changes. Specifically, high-fat diet caused a pronouncedenlargement of the interscapular depot as well as lightening of itscolor from red to pink (FIG. 10). This diet-induced enlargementreflected a doubling of the mass (FIG. 11) and a reduction in thedensity (FIG. 12) of brown fat depots. Depot density was determined bymicro-computed tomography (microCT) in situ for a subset of mice (n=4per group) whose percentages of total body fat, as determined by nuclearmagnetic resonance (NMR), were closest to the group means (all mice werescanned by NMR. In any case, ActRIIB(25-131)-mFc treatment completelyreversed diet-induced changes in brown fat mass (FIG. 11) and density(FIG. 12), while largely reversing diet-induced changes in size andcolor of the depot (FIG. 10). These results indicate that, underhigh-fat dietary conditions, ActRIIB(25-131)-mFc largely or completelyrestores properties likely to correlate with healthy brown fat functionand thus improves the quality of brown fat as it decreases the overallsize of brown fat depots.

Taken together, these data indicate that soluble ActRIIB-Fc fusionproteins can be used as antagonists of signaling by TGF-family ligandsto increase the formation and/or activity of thermogenic brownadiopocytes, and thereby, to treat metabolic conditions exacerbated byhigh caloric intake and potentially other conditions as well.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1-10. (canceled)
 11. A method for increasing thermogenic adipocytes in apatient in need thereof, the method comprising administering aneffective amount of a polypeptide comprising an amino acid sequence thatis at least 95% identical to the sequence of amino acids 29-109 of SEQID NO: 2, wherein administration of the polypeptide increases SIRT-1and/or PGC-1α mRNA levels in adipocytes of the treated patient.
 12. Themethod of claim 11, wherein the polypeptide is a fusion proteincomprising a portion heterologous to ActRIIB.
 13. The method of claim12, wherein the polypeptide is fused to a constant domain of animmunoglobulin.
 14. The method of claim 12, wherein the polypeptide isfused to an Fc portion of an immunoglobulin.
 15. The method of claim 14,wherein the immunoglobulin is a human IgG1.
 16. The method of claim 11,wherein the polypeptide is a dimer.
 17. The method of claim 11, whereinthe polypeptide comprises the sequence of SEQ ID NO:
 5. 18. The methodof claim 11, wherein the polypeptide comprises an amino acid sequencethat is at least 97% identical to the sequence of amino acids 29-109 ofSEQ ID NO:
 2. 19. The method of claim 11, wherein the polypeptidecomprises an amino acid sequence that is at least 90% identical to thesequence of amino acids 25-131 of SEQ ID NO:
 2. 20. The method of claim19, wherein the polypeptide comprises an amino acid sequence that is atleast 95% identical to the sequence of amino acids 25-131 of SEQ ID NO:2.
 21. The method of claim 20, wherein the polypeptide comprises anamino acid sequence that is at least 97% identical to the sequence ofamino acids 25-131 of SEQ ID NO:
 2. 22. The method of claim 11, whereinadministration of the polypeptide increases SIRT-1 mRNA levels inadipocytes of the treated patient.
 23. The method of claim 11, whereinadministration of the polypeptide increases PGC-1α mRNA levels inadipocytes of the treated patient.
 24. The method of claim 11, whereinadministration of the polypeptide increases SIRT-1 and PGC-1α mRNAlevels in adipocytes of the treated patient.
 25. The method of claim 11,wherein administration of the compound promotes uncoupling protein-1(UCP-1) expression in adipocytes of the treated patient.
 26. The methodof claim 11, wherein the polypeptide comprises the sequence of SEQ IDNO: 6.